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US20050079593A1 - Modified enzymes having polymer conjugates - Google Patents

Modified enzymes having polymer conjugates Download PDF

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US20050079593A1
US20050079593A1 US10/623,292 US62329203A US2005079593A1 US 20050079593 A1 US20050079593 A1 US 20050079593A1 US 62329203 A US62329203 A US 62329203A US 2005079593 A1 US2005079593 A1 US 2005079593A1
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Claus von der Osten
Arne Olsen
Erwin Roggen
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Novozymes AS
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Novozymes AS
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    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16—Organic compounds
    • C11D3/38—Products with no well-defined composition, e.g. natural products
    • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16—Organic compounds
    • C11D3/37—Polymers

Definitions

  • the present invention relates to polypeptide-polymer conjugates having added and/or removed one or more attachment groups for coupling polymeric molecules on the surface of the 3D structure of the polypeptide, a method for preparing polypeptide-polymer conjugates of the invention, the use of said conjugated for reducing the immunogenicity and allergenicity, and compositions comprising said conjugate.
  • polypeptides including enzymes
  • the use of polypeptides, including enzymes, in the circulatory system to obtain a particular physiological effect is well-known in the medical arts.
  • industrial applications such as laundry washing, textile bleaching, person care, contact lens cleaning, food and feed preparation enzymes are used as a functional ingredient.
  • the polypeptides are not intended to enter into the circulatory system of the body.
  • Certain polypeptides and enzymes have an unsatisfactory stability and may under certain circumstances—dependent on the way of challenge—cause an immune response, typically an IgG and/or IgE response.
  • GB patent no. 1,183,257 (Crook et al.) describes chemistry for conjugation of enzymes to polysaccharides via a triazine ring.
  • WO 93/15189 (Veronese et al.) concerns a method for maintaining the activity in polyethylene glycol-modified proteolytic enzymes by linking the proteolytic enzyme to a macromolecularized inhibitor.
  • the conjugates are intended for medical applications.
  • EP 183 503 (Beecham Group PLC) discloses a development of the above concept by providing conjugates comprising pharmaceutically useful proteins linked to at least one water-soluble polymer by means of a reversible linking group.
  • EP 471,125 discloses skin care products comprising a parent protease ( Bacillus protease with the trade name Esperase®) coupled to polysaccharides through a triazine ring to improve the thermal and preservation stability.
  • a parent protease Bacillus protease with the trade name Esperase®
  • the coupling technique used is also described in the above mentioned GB patent no. 1,183,257 (Crook et al.).
  • JP 3083908 describes a skin cosmetic material which contains a transglutaminase from guinea pig liver modified with one or more water-soluble substances such as PEG, starch, cellulose etc. The modification is performed by activating the polymeric molecules and coupling them to the enzyme. The composition is stated to be mild to the skin.
  • improved polypeptide-polymer conjugates means in the context of the present invention conjugates having a reduced immune response in humans and animals and/or an improved stability. As will be described further below the immune response is dependent on the way of challenge.
  • polypeptides such as enzymes
  • polypeptides may be made less immunogenic and/or allergenic by adding and/or removing one or more attachment groups on the surface of the parent polypeptide to be coupled to polymeric molecules.
  • the potential risk is an immunogenic response in the form of mainly IgG, IgA and/or IgM antibodies.
  • industrial polypeptides such as enzymes used as a functional ingredient in e.g. detergents, are not intended to enter the circulatory system.
  • the potential risk in connection with industrial polypeptides is inhalation causing an allergenic response in the form of mainly IgE antibody formation.
  • the potential risk is respiratory allergenicity caused by inhalation, intratracheal and intranasal presentation of polypeptides.
  • the main potential risk of pharmaceutical polypeptides is immunogenicity caused by intradermal, intravenous or subcutaneous presentation of the polypeptide.
  • allergic contact dermatitis in a clinical setting and is a cell mediated delayed immune response to chemicals that contact and penetrate the skin.
  • This cell mediated reaction is also termed delayed contact hypersensitivity (type IV reaction according to Gell and Combs classification of immune mechanisms in tissue damage).
  • allergenicity or “respiratory allergenicity” is an immediate anaphylactic reaction (type I antibody-mediated reaction according to Gell and Combs) following inhalation of e.g. polypeptides.
  • polypeptides with a reduced immune response and/or improved stability, which has a substantially retained residual activity.
  • the allergic and the immunogenic response are in one term, at least in the context of the present invention called the “immune response”.
  • the invention relates to a polypeptide-polymer conjugate having
  • parent polypeptide refers to the polypeptide to be modified by coupling to polymeric molecules.
  • the parent polypeptide may be a naturally-occurring (or wild-type) polypeptide or may be a variant thereof prepared by any suitable means.
  • the parent polypeptide may be a variant of a naturally-occurring polypeptide which has been modified by substitution, deletion or truncation of one or more amino acid residues or by addition or insertion of one or more amino acid residues to the amino acid sequence of a naturally-occurring polypeptide.
  • a “suitable attachment group” means in the context of the present invention any amino acid residue group on the surface of the polypeptide capable of coupling to the polymeric molecule in question.
  • Preferred attachment groups are amino groups of lysine residues and the N-terminal amino group.
  • Polymeric molecules may also be coupled to the carboxylic acid groups (—COOH) of amino acid residues in the polypeptide chain located on the surface.
  • Carboxylic acid attachment groups may be the carboxylic acid group of aspartate or glutamate and the C-terminal COOH-group.
  • a “functional site” means any amino acid residues and/or cofactors which are known to be essential for the performance of the polypeptide, such as catalytic activity, e.g. the catalytic triad residues, histidine, aspartate and serine in serine proteases, or e.g. the heme group and the distal and proximal histidines in a peroxidase such as the Arthromyces ramosus peroxidase.
  • catalytic activity e.g. the catalytic triad residues, histidine, aspartate and serine in serine proteases, or e.g. the heme group and the distal and proximal histidines in a peroxidase such as the Arthromyces ramosus peroxidase.
  • the invention relates to a method for preparing improved polypeptide-polymer conjugates comprising the steps of:
  • the invention also relates to the use of a conjugate of the invention and the method of the invention for reducing the immunogenicity of pharmaceuticals and reducing the allergenicity of industrial products.
  • compositions comprising a conjugate of the invention and further ingredients used in industrial products or pharmaceuticals.
  • FIG. 1 shows the anti-lipase serum antibody levels after 5 weekly immunizations with i) control ii) unmodified lipase variant, iii) lipase variant-SPEG. (X: log(serum dilution); Y Optical Density (490/620)).
  • polypeptides used for pharmaceutical applications and industrial application can be quite different the principle of the present invention may be tailored to the specific type of parent polypeptide (i.e. enzyme, hormone peptides etc.).
  • the inventors of the present invention have provided improved polypeptide-polymer conjugates with a reduced immune response in comparison to conjugates prepared from the corresponding parent polypeptides.
  • polypeptides such as enzymes
  • polypeptides may be made less immunogenic and/or less allergenic by adding one or more attachment groups on the surface of the parent polypeptide.
  • the inventors have found that a higher percentage of maintained residual functional activity may be obtained by removing attachment groups at or close to the functional site(s).
  • the invention relates to an improved polypeptide-polymer conjugate having
  • attachment groups should be added and/or removed depends on the specific parent polypeptide.
  • Any available amino acid residues on the surface of the polypeptide may in principle be subject to substitution and/or insertion to provide additional attachment groups.
  • the location of the additional coupled polymeric molecules may be of importance for the reduction of the immune response and the percentage of maintained residual functional activity of the polypeptide itself.
  • a conjugate of the invention may typically have from 1 to 25, preferentially 1 to 10 or more additional polymeric molecules coupled to the surface of the polypeptide in comparison to the number of polymeric molecules of a conjugate prepared on the basis of the corresponding parent polypeptide.
  • the optimal number of attachment groups to be added depends (at least partly) on the surface area (i.e. molecular weight) of the parent polypeptide to be shielded by the coupled polymeric molecules, and also on the number of already available attachment groups on the parent polypeptide.
  • polymeric molecules coupled to the polypeptide might be impeded by the interaction between the polypeptide and its substrate or the like, if they are coupled at or close to the functional site(s) (i.e. active site of enzymes). This will most probably cause reduced activity.
  • conjugates may be constructed to maintain a higher percentage of residual enzymatic activity in comparison to a corresponding conjugates prepared on the basis of the parent enzyme in question. This may be done by substituting and/or deleting attachment groups at or close to the active site, hereby increasing the substrate affinity by improving the accessibility of the substrate in the catalytic cleft.
  • An enzyme-polymer conjugate of the invention may typically have from 1 to 25, preferably 1 to 10 fewer polymeric molecules coupled at or close to the active site in comparison to the number of polymeric molecules of a conjugate prepared on the basis of the corresponding parent polypeptide.
  • the functional site(s) means that no polymeric molecule(s) should be coupled within 5 Angstroms, preferably 8 Angstroms, especially 10 Angstroms of the functional site(s).
  • Removal of attachment groups at or close to the functional site(s) of the polypeptide may advantageously be combined with addition of attachment groups in other parts of the surface of the polypeptide.
  • the total number of attachment groups may this way be unchanged, increased or decreased. However the location(s) of the total number of attachment group(s) is(are) improved assessed by the reduction of the immune response and/or percentage of maintained residual activity. Improved stability may also be obtained this way.
  • the number of attachment groups should be balanced to the molecular weight and/or surface area of the polypeptide. The more heavy the polypeptide is the more polymeric molecules should be coupled to the polypeptide to obtain sufficient shielding of the epitope(s) responsible for antibody formation.
  • the parent polypeptide molecule is relatively light (e.g. 1 to 35 kDa) it may be advantageous to increase the total number of coupled polymeric molecules (outside the functional site(s)) to a total between 4 and 20.
  • the number of coupled polymeric molecules may advantageously be increased to 7 to 40, and so on.
  • the ratio between the molecular weight (Mw) of the polypeptide in question and the number of coupled polymeric molecules considered to be suitable by the inventors is listed below in Table 1.
  • TABLE 1 Molecular weight of parent Number of polymeric molecules polypeptide (M w ) kDa coupled to the polypeptide 1 to 35 4-20 35 to 60 7-40 60 to 80 10-50 80 to 100 15-70 More than 100 more than 20 Reduced Immune Response vs. Maintained Residual Enzymatic Activity
  • the loss of enzymatic activity of enzyme-polymer conjugates might be a consequence of impeded access of the substrate to the active site in the form of spatial hindrance of the substrate by especially bulky and/or heavy polymeric molecules to the catalytic cleft. It might also, at least partly, be caused by disadvantageous minor structural changes of the 3D structure of the enzyme due to the stress made by the coupling of the polymeric molecules.
  • a polypeptide-polymer conjugates of the invention has a substantially maintained functional activity.
  • a “substantially” maintained functional activity is in the context of the present invention defined as an activity which is at least between 20% and 30%, preferably between 30% and 40%, more preferably between 40% and 60%, better from 60% up to 80%, even better from 80% up to about 100%, in comparison to the activity of the conjugates prepared on the basis of corresponding parent polypeptides.
  • polypeptide-polymer conjugates of the invention where no polymeric molecules are coupled at or close to the functional site(s) the residual activity may even be up to 100% or very close thereto. If attachment group(s) of the parent polypeptide is(are) removed from the functional site the activity might even be more than 100% in comparison to modified (i.e. polymer coupled) parent polypeptide conjugate.
  • the polymeric molecules coupled to the surface of the polypeptide in question should be located in a suitable distance from each other.
  • the parent polypeptide is modified in a manner whereby the polymeric molecules are spread broadly over the surface of the polypeptide.
  • the polypeptide in question has enzymatic activity it is preferred to have as few as possible, especially none, polymeric molecules coupled at or close to the area of the active site.
  • “spread broadly over the surface of the polypeptide” means that the available attachment groups are located so that the polymeric molecules shield different parts of the surface, preferably the whole or close to the whole surface area away from the functional site(s), to make sure that epitope(s) are shielded and hereby not recognized by the immune system or its antibodies.
  • the area of antibody-polypeptide interaction typically covers an area of 500 Angstroms 2 , as described by Sheriff et al., 1987, Proc. Natl. Acad. Sci. USA, 84, 8075-8079.
  • 500 Angstroms 2 corresponds to a rectangular box of 25 Angstroms ⁇ 20 Angstroms or a circular region of radius 12.6 Angstroms. Therefore, to prevent binding of antibodies to the epitope(s) to the polypeptide in question it is preferred to have a maximum distance between two attachment groups around 10 Angstroms.
  • amino acid residues which are located in excess of 10 Angstroms away from already available attachment groups are suitable target residues. If two or more attachment groups on the polypeptide are located very close to each other it will in most cases result in that only one polymeric molecule will be coupled. To ensure a minimal loss of functional activity it is preferred not to couple polymeric molecules at or close to the functional site(s). Said distance depends at least partly on the bulkiness of the polymeric molecules to be coupled, as impeded access by the bulky polymeric molecules to the functional site is undesired. Therefore, the more bulky the polymeric molecules are the longer should the distance from the functional site to the coupled polymeric molecules be.
  • polypeptide having coupled polymeric molecules at (a) known epitope(s) recognizable by the immune system or close to said epitope(s) specific mutations at such sites are also considered advantageous according to the invention. If the position of the epitope(s) is(are) unknown it is advantageous to couple several or many polymeric molecules to the polypeptide.
  • said attachment groups are spread broadly over the surface.
  • Virtually all ionized groups such as the amino groups of lysine residues, are located on the surface of the polypeptide molecule (see for instance Thomas E. Creighton, 1993, “Proteins”, W.H. Freeman and Company, New York).
  • the number of readily accessible attachment groups (e.g. amino groups) on a modified or parent polypeptide equals generally seen the number of lysine residues in the primary structure of the polypeptide plus the N-terminus amino group.
  • Polymeric molecules may also be coupled to the carboxylic groups (—COOH) of amino acid residues on the surface of the polypeptide. Therefore, if using carboxylic groups (including the C-terminal group) as attachment groups addition and/or removal of aspartate and glutamate residues may also be suitable according to the invention.
  • carboxylic groups including the C-terminal group
  • addition and/or removal of aspartate and glutamate residues may also be suitable according to the invention.
  • attachment groups such as —SH groups
  • they may be added and/or removed analogously.
  • substitutions are conservative substitutions.
  • the substitution may advantageously be performed at a location having a distance of 5 Angstroms, preferred 8 Angstroms, especially 10 Angstroms from the functional site(s) (active site for enzymes).
  • An example of a suitable conservative substitution to obtain an additional amino attachment group is an arginine to lysine substitution.
  • Examples of conservative substitutions to obtain additional carboxylic attachment groups are aspargine to aspartate/glutamate or glutamine to aspartate/glutamate substitutions.
  • a lysine residue may be substituted with an arginine and so on.
  • polypeptides includes proteins, peptides and/or enzymes for pharmaceutical or industrial applications.
  • the polypeptides in question have a molecular weight in the range between about 1 to 100 kDa, often 15 kDa and 100 kDa.
  • pharmaceutical polypeptides is defined as polypeptides, including peptides, such as peptide hormones, proteins and/or enzymes, being physiologically active when introduced into the circulatory system of the body of humans and/or animals.
  • compositions are potentially immunogenic as they are introduced into the circulatory system.
  • “pharmaceutical polypeptides” contemplated according to the invention include insulin, ACTH, glucagon, somatostatin, somatotropin, thymosin, parathyroid hormone, pigmentary hormones, somatomedin, erythropoietin, luteinizing hormone, chorionic gonadotropin, hypothalmic releasing factors, antidiuretic hormones, thyroid stimulating hormone, relaxin, interferon, thrombopoietin (TPO) and prolactin.
  • Polypeptides used for industrial applications often have an enzymatic activity.
  • Industrial polypeptides e.g. enzymes
  • industrial polypeptides such as enzymes used as ingredients in industrial compositions and/or products, such as detergents and personal care products, including cosmetics, come into direct contact with the circulatory system of the body of humans or animals, as such enzymes (or products comprising such enzymes) are not injected (or the like) into the bloodstream.
  • the potential risk is respiratory allergy (i.e. IgE response) as a consequence of inhalation to polypeptides through the respiratory passage.
  • industrial polypeptides are defined as polypeptides, including peptides, proteins and/or enzymes, which are not intended to be introduced into the circulatory system of the body of humans and/or animals.
  • polypeptides examples include polypeptides, especially enzymes, used in products such as detergents, household article products, agrochemicals, personal care products, such as skin care products, including cosmetics and toiletries, oral and dermal pharmaceuticals, composition use for processing textiles, compositions for hard surface cleaning, and compositions used for manufacturing food and feed etc.
  • Oxidoreductases E.C. 1, “Enzyme Nomenclature, (1992), Academic Press, Inc.), such as laccase and Superoxide dismutase (SOD); Transferases, (E.C. 2), such as transglutaminases (TGases); Hydrolases (E.C. 3), including proteases, especially subtilisins, and lipolytic enzymes; Isomerases (E.C. 5), such as Protein disulfide Isomerases (PDI).
  • Oxidoreductases E.C. 1, “Enzyme Nomenclature, (1992), Academic Press, Inc.), such as laccase and Superoxide dismutase (SOD); Transferases, (E.C. 2), such as transglutaminases (TGases); Hydrolases (E.C. 3), including proteases, especially subtilisins, and lipolytic enzymes; Isomerases (E.C. 5), such as Protein disulfide Isomerases (PDI).
  • Contemplated proteolytic enzymes include proteases selected from the group of Aspartic proteases, such pepsins, cysteine proteases, such as papain, serine proteases, such as subtilisins, or metallo proteases, such as NEUTRASE®.
  • parent proteases include PD498 (WO 93/24623 and SEQ ID NO: 2), SAVINASE® (von der Osten et al., 1993, Journal of Biotechnology, 28, 55+, SEQ ID NO: 3), Proteinase K (Gunkel et al., 1989, Eur. J. Biochem, 179, 185-194), Proteinase R (Samal et al, 1990, Mol. Microbiol, 4, 1789-1792), Proteinase T (Samal et al., 1989, Gene, 85, p. 329-333), Subtilisin DY (Betzel et al. 1993, Arch.
  • Contemplated lipolytic enzymes include Humicola lanuginosa lipases, e.g. the one described in EP 258 068 and EP 305 216 (See SEQ ID NO: 6 below), Humicola insolens , a Rhizomucor miehei lipase, e.g. as described in EP 238 023, Absidia sp. lipolytic enzymes (WO 96/13578), a Candida lipase, such as a C. antarctica lipase, e.g. the C. antarctica lipase A or B described in EP 214 761, a Pseudomonas lipase such as a P. alcaligenes and P.
  • Humicola lanuginosa lipases e.g. the one described in EP 258 068 and EP 305 216 (See SEQ ID NO: 6 below
  • Humicola insolens e.g. as described in EP 238 023
  • pseudoalcaligenes lipase e.g. as described in EP 218 272
  • a P. cepacia lipase e.g. as described in EP 331 376
  • a Pseudomonas sp. lipase as disclosed in WO 95/14783
  • Bacillus lipase e.g. a B. subtilis lipase (Dartois et al., 1993 Biochemica et Biophysica Acta 1131, 253-260)
  • a B. stearothermophilus lipase JP 64/744992
  • a B. pumilus lipase WO 91/16422
  • lipolytic enzymes include cutinases, e.g. derived from Pseudomonas mendocina as described in WO 88/09367, or a cutinase derived from Fusarium solani pisi (e.g. described in WO 90/09446).
  • Contemplated laccases include Polyporus pinisitus laccase (WO 96/00290), Myceliophthora laccase (WO 95/33836), Scytalidium laccase (WO 95/338337), and Pyricularia oryzae laccase (Available from Sigma).
  • Contemplated peroxidases include B. pumilus peroxidases (WO 91/05858), Myxococcaceae peroxidase (WO 95/11964), Coprinus cinereus (WO 95/10602) and Arthromyces ramosus peroxidase (Kunishima et al. 1994, J. Mol. Biol., 235, 331-344).
  • Suitable transferases include any transglutaminases disclosed in WO 96/06931 (Novo Nordisk A/S) and WO 96/22366 (Novo Nordisk A/S).
  • suitable protein disulfide isomerases include PDIs described in WO 95/01425 (Novo Nordisk A/S).
  • the polymeric molecules coupled to the polypeptide may be any suitable polymeric molecule, including natural and synthetic homo-polymers, such as polyols (i.e. poly-OH), polyamines (i.e. poly-NH 2 ) and polycarboxyl acids (i.e. poly-COOH), and further hetero-polymers i.e. polymers comprising one or more different coupling groups e.g. a hydroxyl group and amine groups.
  • polyols i.e. poly-OH
  • polyamines i.e. poly-NH 2
  • polycarboxyl acids i.e. poly-COOH
  • hetero-polymers i.e. polymers comprising one or more different coupling groups e.g. a hydroxyl group and amine groups.
  • polymeric molecules include polymeric molecules selected from the group comprising polyalkylene oxides (PAO), such as polyalkylene glycols (PAG), including polyethylene glycols (PEG), methoxypolyethylene glycols (mPEG) and polypropylene glycols, PEG-glycidyl ethers (Epox-PEG), PEG-oxycarbonylimidazole (CDI-PEG), branched PEGs, polyvinyl alcohol (PVA), polycarboxylates, polyvinylpyrolidones, poly-D,L-amino acids, polyethylene-co-maleic acid anhydride, polystyrene-co-malic acid anhydride, dextrans including carboxymethyl-dextrans, heparin, homologous albumin, celluloses, including methylcellulose, carboxymethylcellulose, ethylcellulose, hydroxyethylcellulose carboxyethylcellulose and hydroxypropylcellulose, hydrolysates of chi
  • Preferred polymeric molecules are non-toxic polymeric molecules such as (m)polyethylene glycol ((m)PEG) which further requires a relatively simple chemistry for its covalently coupling to attachment groups on the enzyme's surface.
  • polyalkylene oxides such as polyethylene oxides, such as PEG and especially mPEG
  • PEO polyalkylene oxides
  • mPEG polyethylene oxides
  • PEG and especially mPEG are the preferred polymeric molecules, as these polymeric molecules, in comparison to polysaccharides such as dextran, pullulan and the like, have few reactive groups capable of cross-linking.
  • methoxypolyethylene glycols may advantageously be used. This arises from the fact that methoxyethylene glycols have only one reactive end capable of conjugating with the enzyme. Consequently, the risk of cross-linking is less pronounced. Further, it makes the product more homogeneous and the reaction of the polymeric molecules with the enzyme easier to control.
  • Enzyme variants to be conjugated may be constructed by any suitable method. A number of methods are well established in the art. For instance enzyme variants according to the invention may be generated using the same materials and methods described in e.g. WO 89/06279 (Novo Nordisk A/S), EP 130,756 (Genentech), EP 479,870 (Novo Nordisk A/S), EP 214,435 (Henkel), WO 87/04461 (Amgen), WO 87/05050 (Genex), EP application no.
  • the gene encoding the polypeptide of interest Prior to mutagenesis the gene encoding the polypeptide of interest must be cloned in a suitable vector. Methods for generating mutations in specific sites are described below.
  • these mutations can be introduced using synthetic oligonucleotides. These oligonucleotides contain nucleotide sequences flanking the desired mutation sites; mutant nucleotides are inserted during oligo-nucleotide synthesis.
  • Site-directed mutagenesis is carried out by SOE-PCR mutagenesis technique described by Kammann et al., 1989, Nucleic Acids Research, 17(13), 5404, and by Sarkar G. and Sommer, S. S., 1990, Biotechniques, 8, 404-407.
  • polymeric molecules to be conjugated with the polypeptide in question are not active it must be activated by the use of a suitable technique. It is also contemplated according to the invention to couple the polymeric molecules to the polypeptide through a linker. Suitable linkers are well-known to the skilled person.
  • Some of the methods concern activation of insoluble polymers but are also applicable to activation of soluble polymers e.g. periodate, trichlorotriazine, sulfonylhalides, divinylsulfone, carbodiimide etc.
  • the functional groups being amino, hydroxyl, thiol, carboxyl, aldehyde or sulfydryl on the polymer and the chosen attachment group on the protein must be considered in choosing the activation and conjugation chemistry which normally consist of i) activation of polymer, ii) conjugation, and iii) blocking of residual active groups.
  • Coupling polymeric molecules to the free acid groups of polypeptides may be performed with the aid of diimide and for example amino-PEG or hydrazino-PEG (Pollak et al., 1976, J. Amr. Chem. Soc., 98, 289-291) or diazoacetate/amide (Wong et al., 1992, “Chemistry of Protein Conjugation and Crosslinking”, CRC Press).
  • Coupling polymeric molecules to hydroxy groups are generally very difficult as it must be performed in water. Usually hydrolysis predominates over reaction with hydroxyl groups.
  • Coupling polymeric molecules to free sulfhydryl groups can be reached with special groups like maleimido or the ortho-pyridyl disulfide.
  • vinylsulfone U.S. Pat. No. 5,414,135, (1995), Snow et al.
  • Accessible arginine residues in the polypeptide chain may be targeted by groups comprising two vicinal carbonyl groups.
  • Organic sulfonyl chlorides e.g. tresyl chloride
  • hydroxy groups in a number of polymers e.g. PEG
  • good leaving groups sulfonates
  • nucleophiles like amino groups in polypeptides allow stable linkages to be formed between polymer and polypeptide.
  • the reaction conditions are in general mild (neutral or slightly alkaline pH, to avoid denaturation and little or no disruption of activity), and satisfy the non-destructive requirements to the polypeptide.
  • Tosylate is more reactive than the mesylate but also more unstable decomposing into PEG, dioxane, and sulfonic acid (Zalipsky, 1995, Bioconjugate Chem., 6, 150-165). Epoxides may also been used for creating amine bonds but are much less reactive than the above mentioned groups.
  • isocyanates and isothiocyanates may be employed yielding ureas and thioureas, respectively.
  • Amides may be obtained from PEG acids using the same leaving groups as mentioned above and cyclic imide thrones (U.S. Pat. No. 5,349,001 (1994), Greenwald et al.). The reactivity of these compounds is very high but may make the hydrolysis to fast.
  • PEG succinate made from reaction with succinic anhydride can also be used.
  • the hereby comprised ester group make the conjugate much more susceptible to hydrolysis (U.S. Pat. No. 5,122,614, 1992, Zalipsky). This group may be activated with N-hydroxy succinimide.
  • Coupling of PEG to an aromatic amine followed by diazotization yields a very reactive diazonium salt which in situ can be reacted with a peptide.
  • An amide linkage may also be obtained by reacting an azlactone derivative of PEG (U.S. Pat. No. 5,321,095, 1994, Greenwald, R. B.) thus introducing an additional amide linkage.
  • peptides do not comprise many lysines, it may be advantageous to attach more than one PEG to the same lysine. This can be done e.g. by the use of 1,3-diamino-2-propanol.
  • PEGs may also be attached to the amino-groups of the enzyme with carbamate linkages (WO 95/11924, Greenwald et al.). Lysine residues may also be used as the backbone.
  • the coupling technique used in the examples is the N-succinimidyl carbonate conjugation technique described in WO 90/13590 (Enzon).
  • a 3-dimensional structure of the parent polypeptide in question is required.
  • This structure may for example be an X-ray structure, an NMR structure or a model-built structure.
  • the Brookhaven Databank is a source of X-ray- and NMR-structures.
  • a model-built structure may be produced by the person skilled in the art if one or more 3D-structure(s) exist(s) of homologous polypeptide(s) sharing at least 30% sequence identity with the polypeptide in question.
  • 3D-structure(s) may be produced by the person skilled in the art if one or more 3D-structure(s) exist(s) of homologous polypeptide(s) sharing at least 30% sequence identity with the polypeptide in question.
  • Several software packages exist which may be employed to construct a model structure One example is the Homology 95.0 package from Biosym.
  • Typical actions required for the construction of a model structure are: alignment of homologous sequences for which 3D-structures exist, definition of Structurally conserveed Regions (SCRs), assignment of coordinates to SCRs, search for structural fragments/loops in structure databases to replace Variable Regions, assignment of coordinates to these regions, and structural refinement by energy minimization. Regions containing large inserts (>3 residues) relative to the known 3D-structures are known to be quite difficult to model, and structural predictions must be considered with care.
  • this structure serves as an essential prerequisite for the fulfillment of the method described below.
  • Target amino acid residues to be mutated are according to the invention selected in order to obtain additional or fewer attachment groups, such as free amino groups (—NH 2 ) or free carboxylic acid groups (—COOH), on the surface of the polypeptide and/or to obtain a more complete and broadly spread shielding of the epitope(s) on the surface of the polypeptide.
  • attachment groups such as free amino groups (—NH 2 ) or free carboxylic acid groups (—COOH
  • this may be done by substitution of arginine to lysine, which are both positively charged, but only the lysine having a free amino group suitable as an attachment group.
  • the conservative substitution may for instance be an aspargine to aspartic acid or glutamine to glutamic acid substitution. These residues resemble each other in size and shape, except from the carboxylic groups being present on the acidic residues.
  • a lysine may be substituted with an arginine, and so on.
  • the mutation may also be on target amino acid residues which are less/non-conservative. Such mutation is suitable for obtaining a more complete and broadly spread shielding of the polypeptide surface than can be obtained by the conservative substitutions.
  • Attachment_residue residue(s) which can bind polymeric molecules, e.g. lysines (amino group) or aspartic/glutamic acids (carboxylic groups). N- or C-terminal amino/carboxylic groups are to be included where relevant.
  • Mutation_residue residue(s) which is to be mutated, e.g. arginine or aspargine/glutamine.
  • Essential_catalytic_residues residues which are known to be essential for catalytic function, e.g. the catalytic triad in serine proteases.
  • Solvent_exposed_residues are defined as residues which are at least 5% exposed according to the BIOSYM/INSIGHT algorithm found in the module Homology 95.0.
  • the file filename_area.tab is produced. Note: For this program to function properly all water molecules must first be removed from the structure.
  • step c) The mutation(s) performed in step c) may be performed by standard techniques well known in the art, such as site-directed mutagenesis (see, e.g., Sambrook et al., 1989, Molecular Cloning. A Laboratory Manual, Cold Spring Harbor, N.Y.
  • nucleotide substitution can be found in e.g. Ford et al., 1991, Protein Expression and Purification, 2, 95-107.
  • Polypeptide-polymer conjugates of the invention may be prepared by any coupling method known in the art including the above mentioned techniques.
  • polymeric molecules to be conjugated with the polypeptide are not active it must be activated by the use of a suitable method.
  • the polymeric molecules may be coupled to the polypeptide through a linker. Suitable linkers are well known to the skilled person.
  • Some of the methods concern activation of insoluble polymers but are also applicable to activation of soluble polymers e.g. periodate, trichlorotriazine, sulfonylhalides, divinylsulfone, carbodiimide etc.
  • the functional groups being amino, hydroxyl, thiol, carboxyl, aldehyde or sulfydryl on the polymer and the chosen attachment group on the protein must be considered in choosing the activation and conjugation chemistry which normally consists of i) activation of polymer, ii) conjugation, and iii) blocking of residual active groups.
  • Coupling polymeric molecules to the free acid groups of enzymes can be performed with the aid of diimide and for example amino-PEG or hydrazino-PEG (Pollak et al., 1976, J. Amr. Chem. Soc., 98, 289-291) or diazoacetate/amide (Wong et al., 1992, “Chemistry of Protein Conjugation and Crosslinking”, CRC Press).
  • Coupling polymeric molecules to hydroxy groups are generally very difficult as it must be performed in water. Usually hydrolysis predominates over reaction with hydroxyl groups.
  • Coupling polymeric molecules to free sulfhydryl groups can be reached wih special groups like maleimido or the ortho-pyridyl disulfide.
  • vinylsulfone U.S. Pat. No. 5,414,135 (1995), Snow et al.
  • Accessible arginine residues in the polypeptide chain may be targeted by groups comprising two vicinal carbonyl groups.
  • Organic sulfonyl chlorides e.g. tresyl chloride
  • hydroxy groups in a number of polymers e.g. PEG
  • good leaving groups sulfonates
  • nucleophiles like amino groups in polypeptides allow stable linkages to be formed between polymer and polypeptide.
  • the reaction conditions are in general mild (neutral or slightly alkaline pH, to avoid denaturation and little or no disruption of activity), and satisfy the non-destructive requirements to the polypeptide.
  • Tosylate is more reactive than the mesylate but also more unstable decomposing into PEG, dioxane, and sulfonic acid (Zalipsky, 1995, Bioconjugate Chem., 6, 150-165). Epoxides may also been used for creating amine bonds but are much less reactive than the above mentioned groups.
  • isocyanates and isothiocyanates may be employed yielding ureas and thioureas, respectively.
  • Amides may be obtained from PEG acids using the same leaving groups as mentioned above and cyclic imide thrones (U.S. Pat. No. 5,349,001 (1994), Greenwald et al.). The reactivity of these compounds is very high but may make the hydrolysis to fast.
  • PEG succinate made from reaction with succinic anhydride can also be used.
  • the hereby comprised ester group makes the conjugate much more susceptible to hydrolysis (U.S. Pat. No. 5,122,614, (1992), Zalipsky). This group may be activated with N-hydroxy succinimide.
  • Coupling of PEG to an aromatic amine followed by diazotization yields a very reactive diazonium salt which in situ can be reacted with a peptide.
  • An amide linkage may also be obtained by reacting an azlactone derivative of PEG (U.S. Pat. No. 5,321,095, (1994), Greenwald, R. B.) thus introducing an additional amide linkage.
  • peptides do not comprise many lysines, it may be advantageous to attach more than one PEG to the same lysine. This can be done e.g. by the use of 1,3-diamino-2-propanol.
  • PEGs may also be attached to the amino-groups of the enzyme with carbamate linkages (WO 95/11924, Greenwald et al.). Lysine residues may also be used as the backbone.
  • a specific example of a protease is the parent PD498 (WO 93/24623 and SEQ ID NO: 2).
  • the parent PD498 has a molecular weight of 29 kDa.
  • Lysine and arginine residues are located as follows: Distance from the active site Arginine Lysine 0-5 Angstroms 1 5-10 Angstroms 10-15 Angstroms 5 6 15-20 Angstroms 2 3 20-25 Angstroms 1 3 Total 9 12
  • Suitable conservative arginine to lysine substitutions in parent PD498 may be any of R51K, R62K, R121K, R169K, R250K, R28K, R190K.
  • Suitable non-conservative substitutions in parent PD498 may be any of P6K, Y7K, S9K, A10K, Y11K, Q12K, D43K, Y44K, N45K, N65K, G87K, 188K, N209K, A211K, N216K, N217K, G218K, Y219K, S220K, Y221K, G262K.
  • PD498 variant-SPEG conjugates may be prepared using any of the above mentioned PD498 variants as the starting material by any conjugation technique known in the art for coupling polymeric molecules to amino groups on the enzyme. A specific example is described below.
  • BPN′ A specific example of a protease having an attachment group in the active site is BPN′ which has 11 attachment groups (plus an N-terminal amino group): BPN′ has a molecular weight of 28 kDa.
  • Lysine and arginine residues are located as follows: Distance from the active site Arginine Lysine 0-5 Angstroms 1 5-10 Angstroms 10-15 Angstroms 1 4 15-20 Angstroms 1 4 20-25 Angstroms 2 Total 2 11
  • the lysine residue located within 0-5 Angstroms of the active site can according to the invention advantageously be removed. Specifically this may be done by a K94R substitution.
  • BPN′ variant-SPEG conjugates may be prepared using the above mentioned BPN′ variant as the starting material by any conjugation technique known in the art for coupling polymeric molecules to amino groups on the enzyme.
  • SAVINASE® von der Osten et al., 1993, Journal of Biotechnology, 28, 55+ and SEQ ID NO: 3 may according to the invention have added a number of amino attachment groups to the surface and removed an amino attachment group close to the active site.
  • substitutions in SAVINASE® are sites for mutagenesis: R10K, R19K, R45K, R145K, R170K, R186K and R247K.
  • substitution K94R is identified as a mutation suitable for preventing attachment of polymers close to active site.
  • SAVINASE® variant-SPEG conjugates may be prepared using any of the above mentioned SAVINASE® variants as the starting material by any conjugation technique known in the art for coupling polymeric molecules to amino groups on the enzyme.
  • lipase variants with reduced immunogenicity using the parent Huminocal lanuginosa DSM 4109 lipase see SEQ ID NO: 6 as the backbone for substitutions are listed below.
  • the parent unmodified Humicola lanuginosa lipase has 8 attachment groups including the N-terminal NH 2 group and a molecular weight of about 29 kDa.
  • Suitable conservative arginine to lysine substitutions in the parent lipase may be any of R133K, R139K, R160K, R179K, R209K, R118K and R125K.
  • Suitable non-conservative substitutions in the parent lipase may be any of: A18K, G31K, T32K, N33K, G38K, A40K, D48K, T50K, E56K, D57K, S58K, G59K, V60K, G61K, D62K, T64K, L78K, N88K, G91K, N92K, L93K, S105K, G106K, V120K, P136K, G225K, L227K, V228K, P229K, P250K, F262K.
  • Non-conservative substitution in the Humicola lanuginosa lipase include: E87K or D254K.
  • Lipase variant-SPEG conjugates may be prepared using any of the above mentioned lipase variants as the starting material by any conjugation technique known in the art for coupling polymeric molecules to amino groups on the enzyme. A specific example is described below.
  • Example 12 it is shown that a conjugate of the Humicola lanuginosa lipase variant with E87K + D254K substitutions coupled to S-PEG 15,000 has reduced immunogenic response in Balb/C mice in comparison to the corresponding parent unmodified enzyme.
  • Immunogenicity is a broader term than “antigenicity” and “allergenicity”, and expresses the immune system's response to the presence of foreign substances. Said foreign substances are called immunogens, antigens and allergens depending of the type of immune response they elicit.
  • immunoglobulin may be defined as a substance which, when introduced into circulatory system of animals and humans, is capable of stimulating an immunologic response resulting in formation of immunoglobulin.
  • antigen refers to substances which by themselves are capable of generating antibodies when recognized as a non-self molecule.
  • an “allergen” may be defined as an antigen which may give rise to allergic sensitization or an allergic response by IgE antibodies (in humans, and molecules with comparable effects in animals).
  • Assessment of the immunogenicity may be made by injecting animal subcutaneously to enter the immunogen into the circulation system and comparing the response with the response of the corresponding parent polypeptide.
  • the “circulatory system” of the body of humans and animals means, in the context of the present invention, the system which mainly consists of the heart and blood vessels.
  • the heart delivers the necessary energy for maintaining blood circulation in the vascular system.
  • the circulation system functions as the organism's transportation system, when the blood transports O 2 , nutritious matter, hormones, and other substances of importance for the cell regulation into the tissue. Further the blood removes CO 2 from the tissue to the lungs and residual substances to e.g. the kidneys. Furthermore, the blood is of importance for the temperature regulation and the defense mechanisms of the body, which include the immune system.
  • This model seeks to identify the immunogenic response in the form of the IgG response in Balb/C mice being injected subcutaneously with modified and unmodified polypeptides.
  • animal models can be used for assessment of the immunogenic potential.
  • a polypeptide having “reduced immunogenicity” according to the invention indicates that the amount of produced antibodies, e.g. immunoglobulin in humans, and molecules with comparable effects in specific animals, which can lead to an immune response, is significantly decreased, when introduced into the circulatory system, in comparison to the corresponding parent polypeptide.
  • mice For Balb/C mice the IgG response gives a good indication of the immunigenic potential of polypeptides.
  • Assessment of allergenicity may be made by inhalation tests, comparing the effect of intratracheally (into the trachea) administrated parent enzymes with the corresponding modified enzymes according to the invention.
  • a suitable strain of guinea pigs does not as humans, produce IgE antibodies in connection with the allergic response. However, they produce another type of antibody the IgG1A and IgG1B (see e.g. Prent ⁇ , ATLA, 19, 8-14, 1991), which are responsible for their allergenic response to inhaled polypeptides including enzymes. Therefore, when using the Dunkin Hartley animal model, the relative amount of IgG1A and IgG1B is a measure of the allergenicity level.
  • Balb/C mice strain is suitable for intratracheal exposure.
  • Balb/C mice produce IgE as the allergic response.
  • mice such as rats, rabbits etc. may also be used for comparable studies.
  • the invention relates to a composition comprising a polypeptide-polymer conjugate of the invention.
  • the composition may be a pharmaceutical or industrial composition.
  • the composition may further comprise other polypeptides, proteins or enzymes and/or ingredients normally used in e.g. detergents, including soap bars, household articles, agrochemicals, personal care products, including skin care compositions, cleaning compositions for e.g. contact lenses, oral and dermal pharmaceuticals, composition use for treating textiles, compositions used for manufacturing food, e.g. baking, and feed etc.
  • detergents including soap bars, household articles, agrochemicals, personal care products, including skin care compositions, cleaning compositions for e.g. contact lenses, oral and dermal pharmaceuticals, composition use for treating textiles, compositions used for manufacturing food, e.g. baking, and feed etc.
  • the invention also relates to the use of the method of the invention for reducing the immune response of polypeptides.
  • industrial products such as detergents, such as laundry, dish wash and hard surface cleaning detergents, and food or feed products.
  • B. subtilis 309 and 147 are variants of Bacillus lentus , deposited with the NCIB and accorded the accession numbers NCIB 10309 and 10147, and described in U.S. Pat. No. 3,723,250 incorporated by reference herein.
  • E. coli MC 1000 (M. J. Casadaban and S. N. Cohen (1980); J. Mol. Biol. 138 179-207), was made r ⁇ ,m + by conventional methods and is also described in US Patent Application Serial No. 039,298.
  • pPD498 E. coli - B. subtilis shuttle vector (described in U.S. Pat. No. 5,621,089 under section 6.2.1.6) containing the wild-type gene encoding for PD498 protease (SEQ ID NO: 2). The same vector is used for mutagenesis in E. coli as well as for expression in B. subtilis.
  • Horse Radish Peroxidase labeled anti-rat-Ig (Dako, DK, P162, # 031; dilution 1:1000).
  • Mouse anti-rat IgE (Serotec MCA193; dilution 1:200).
  • Rat anti-mouse IgE (Serotec MCA419; dilution 1:100).
  • Biotin-labeled mouse anti-rat IgG1 monoclonal antibody (Zymed 03-9140; dilution 1:1000)
  • Biotin-labeled rat anti-mouse IgG1 monoclonal antibody (Serotec MCA336B; dilution 1:1000)
  • Rat anti-Mouse IgG1, biotin (SeroTec, Cat# MCA336B)
  • Ortho-Phenylene-diamine (Kem-en-Tec)
  • Preactivated plates can be stored at room temperature for 3 weeks when kept in a plastic bag.
  • ELISA microtiter plates are coated with rabbit anti-PD498 1:8000 in carbonate buffer and incubated overnight at 4° C. The next day the plates are blocked with 2% BSA for 1 hour and washed 3 times with PBS Tween 20.
  • Alkaline phosphatase marked rabbit anti-goat 1:8000 (Sigma A4187) is applied and incubated for 1 hour, washed 2 times in PBS Tween20 and 1 time with diethanol amine buffer.
  • the marked alkaline phosphatase is developed using p-nitrophenyl phosphate for 30 minutes at 37° C. or until appropriate color has developed.
  • the reaction is stopped using stop medium (K 2 HPO 4 /HaH 3 buffer comprising EDTA (pH 10)) and read at OD 405/650 using an ELISA reader.
  • stop medium K 2 HPO 4 /HaH 3 buffer comprising EDTA (pH 10)
  • Double blinds are included on all ELISA plates.
  • Electrophoretic separation of proteins was performed by standard methods using 4-20% gradient SDS poly acrylamide gels (Novex). Proteins were detected by silver staining. The molecule weight was measured relative to the mobility of Mark-12@ wide range molecule weight standards from Novex.
  • Proteases cleave the bond between the peptide and p-nitroaniline to give a visible yellow color absorbing at 405 nm.
  • Buffer e.g. Britton and Robinson buffer pH 8.3.
  • Substrate 100 mg suc-AAPF-pNa is dissolved into 1 ml dimethyl sulfoxide (DMSO). 100 microliters of this is diluted into 10 ml with Britton and Robinson buffer.
  • DMSO dimethyl sulfoxide
  • the substrate and protease solution is mixed and the absorbance is monitored at 405 nm as a function of time and ABS 405 nm /min.
  • the temperature should be controlled (20-50° C. depending on protease). This is a measure of the protease activity in the sample.
  • proteolytic activity is expressed in Kilo NOVO Protease Units (KNPU).
  • KNPU Kilo NOVO Protease Units
  • the activity is determined relatively to an enzyme standard (SAVINASE®), and the determination is based on the digestion of a dimethyl casein (DMC) solution by the proteolytic enzyme at standard conditions, i.e. 50° C., pH 8.3, 9 min. reaction time, 3 min. measuring time.
  • DMC dimethyl casein
  • a folder AF 220/1 is available upon request to Novo Nordisk A/S, Denmark, which folder is hereby included by reference.
  • a Glycine Unit is defined as the proteolytic enzyme activity which, under standard conditions, during a 15-minute incubation at 40° C., with N-acetyl casein as substrate, produces an amount of NH 2 -group equivalent to 1 mmole of glycine.
  • Enzyme activity can also be measured using the PNA assay, according to reaction with the soluble substrate succinyl-alanine-alanine-proline-phenyl-alanine-para-nitrophenol, which is described in Rothgeb, T. M., Goodlander, B. D., Garrison, P. H., and Smith, L. A., 1988 Journal of American Oil Chemists Society.
  • Fermentation of PD498 variants in B. subtilis are performed at 30° C. on a rotary shaking table (300 r.p.m.) in 500 ml baffled Erlenmeyer flasks containing 100 ml BPX medium for 5 days. In order to make an e.g. 2 liter broth 20 Erlenmeyer flasks are fermented simultaneously.
  • BPX Composition (per liter) Potato starch 100 g Ground barley 50 g Soybean flour 20 g Na 2 HPO 4 ⁇ 12 H 2 O 9 g Pluronic 0.1 g Sodium caseinate 10 g
  • the starch in the medium is liquefied with alpha-amylase and the medium is sterilized by heating at 120° C. for 45 minutes. After sterilization the pH of the medium is adjusted to 9 by addition of NaHCO 3 to 0.1 M.
  • Approximately 1.6 liters of PD498 variant fermentation broth are centrifuged at 5000 rpm for 35 minutes in 1 liter beakers.
  • the supernatants are adjusted to pH 7.0 using 10% acetic acid and filtered on Seitz Supra S100 filter plates.
  • the filtrates are concentrated to approximately 400 ml using an Amicon CH2A UF unit equipped with an Amicon S1Y10 UF cartridge.
  • the UF concentrate is centrifuged and filtered prior to absorption at room temperature on a Bacitracin affinity column at pH 7.
  • the PD498 variant is eluted from the Bacitracin column at room temperature using 25% 2-propanol and 1 M sodium chloride in a buffer solution with 0.01 dimethylglutaric acid, 0.1 M boric acid and 0.002 M calcium chloride adjusted to pH 7.
  • fractions with protease activity from the Bacitracin purification step are combined and applied to a 750 ml Sephadex G25 column (5 cm diameter) equilibrated with a buffer containing 0.01 dimethylglutaric acid, 0.1 M boric acid and 0.002 M calcium chloride adjusted to pH 6.0.
  • Fractions with proteolytic activity from the Sephadex G25 column are combined and applied to a 150 ml CM Sepharose CL 6B cation exchange column (5 cm diameter) equilibrated with a buffer containing 0.01 M dimethylglutaric acid, 0.1 M boric acid, and 0.002 M calcium chloride adjusted to pH 6.0.
  • the protease is eluted using a linear gradient of 0-0.5 M sodium chloride in 1 liter of the same buffer.
  • Protease containing fractions from the CM Sepharose column are combined and filtered through a 2 micron filter.
  • the antigen is diluted to 1 mg/ml in carbonate buffer.
  • the plates are coated overnight at 4° C.
  • Unspecific adsorption is blocked by incubating each well for 1 hour at room temperature with 200 ml blocking buffer.
  • the plates are washed 3 ⁇ with 300 ml washing buffer.
  • Unknown mouse sera are diluted in dilution buffer, typically 10 ⁇ , 20 ⁇ and 40 ⁇ , or higher.
  • Unbound material is removed by washing 3 ⁇ with washing buffer.
  • the anti-Mouse IgG1 antibody is diluted 2000 ⁇ in dilution buffer.
  • Unbound material is removed by washing 3 ⁇ with washing buffer.
  • Streptavidine is diluted 1000 ⁇ in dilution buffer.
  • Unbound material is removed by washing 3 ⁇ with 300 ml washing buffer.
  • OPD 0.6 mg/ml
  • H 2 O 2 0.4 ml/ml
  • the reaction is stopped by adding 100 ml H 2 SO 4 .
  • the plates are read at 492 nm with 620 nm as reference.
  • Balb/C mice (20 grams) are immunized 10 times (intervals of 14 days) by subcutaneous injection of the modified or unmodified polypeptide in question, respectively by standard procedures known in art.
  • the 3D structure of parent PD498 was modeled as described above based on 59% sequence identity with Thermitase® (2tec.pdb).
  • the sequence of PD498 is SEQ ID NO: 2.
  • PD498 residue numbering is used, 1-280.
  • Lines 1-8 The subset ALLZONE is defined as those residues which are either within 10 Angstroms of the free amino groups on lysines or the N-terminal, or within 8 Angstroms of the catalytic triad residues 39, 72 and 226.
  • Line 10 The subset REST is defined as those residues not included in ALLZONE.
  • Subset SUB5B is defined as those residues in a 5 Angstroms shell around REST, excluding residues within 8 Angstroms of the catalytic residues.
  • REST contains Arg62 and Arg169
  • SUB5B contains Arg51, Arg121, and Arg250.
  • ACTSITE contains Arg103, but position 103 is within 8 Angstroms from essential_catalytic_residues, and thus not relevant.
  • substitutions R51K, R62K, R121K, R169K and R250K are identified in parent PD498 as suitable sites for mutagenesis.
  • the residues are substituted below in section 2, and further analysis done:
  • Lines 1-15 Solvent exposed arginines in subsets REST and SUB5B are replaced by lysines. Solvent accessibilities are recalculated following arginine replacement.
  • Lines 16-23 The subset ALLZONEx is defined as those residues which are either within 10 Angstroms of the free amino groups on lysines (after replacement) or the N-terminal, or within 8 Angstroms of the catalytic triad residues 39, 72 and 226.
  • SAVINASE® The 3D structure of SAVINASE® is available in the Brookhaven Databank as lsvn.pbd.
  • a related subtilisin is available as 1st3.pdb.
  • SAVINASE® The sequence of SAVINASE® is shown in SEQ ID NO: 3.
  • the sequence numbering used is that of subtilisin BPN′, SAVINASE® having deletions relative to BPN′ at positions 36, 56, 158-159 and 163-164.
  • the active site residues (functional site) are D32, H64 and S221.
  • SAVINASE® REST contains the arginines Arg10, Arg170 and Arg 186, and SUB5B contains Arg19, Arg45, Arg145 and Arg247.
  • the substitution K94R is a mutation removing lysine as attachment group close to the active site.
  • SAVINASE® P5K, P14K, T22K, T38K, H39K, P40K, L42K, L75K, N76K, L82K, P86K, S103K, V104K, S105K, A108K, A133K, T134K, L135K, Q137K, N140K, N173K, N204K, Q206K, G211K, S212K, T213K, A215K, S216K, N269K.
  • the subset REST contains Gln33 and Asn245, SUB5B contains Gln12, Gln126, Asn209, Gln242, Asn246, Gln248 and Asn266, all of which are solvent exposed.
  • substitutions Q12E or Q12D, Q33E or Q33D, Q126E or Q126D, N209D or N209E, Q242E or Q242D, N245D or N245E, N246D or N246E, Q248E or Q248D and N266D or N266E are identified in PD498 as sites for mutagenesis within the scope of this invention. Residues are substituted below in section 2, and further analysis done:
  • the subset RESTx contains only two residues: A233 and G234, none of which are solvent exposed. No further mutagenesis is required to obtain complete protection of the surface. However, it may be necessary to remove some of the reactive carboxylic groups in the active site region to ensure access to the active site of PD498. Acidic residues within the subset ACTSITE are: D39, D58, D68 and D106. Of these only the two latter are solvent exposed and D39 is a functional residue. The mutations D68N, D68Q, D106N and D106Q were found suitable according to the present invention.
  • Arthromyces ramosus peroxidase Suitable substitutions in the Arthromyces ramosus peroxidase for addition of carboxylic acid attachment groups (—COOH) Suitable locations for addition of carboxylic attachment groups (aspartatic acids and glutamic acids) in a non-hydrolytic enzyme, Arthromyces ramosus peroxidase were found as follows.
  • This oxido-reductase contains 344 amino acid residues. The first eight residues are not visible in the X-ray structure: QGPGGGGG, and N143 is glycosylated.
  • the commands performed in Insight are shown in the command files makeDEzone.bcl and makeDEzone2.bcl below.
  • the C-terminal residue is P344, the ACTSITE is defined as the heme group and the two histidines coordinating it (H56 & H184).
  • the subset REST contains Gln70, and SUB5B contains Gln34, Asn128, Asn303 all of which are solvent exposed.
  • the substitutions Q34E or Q34D, Q70E or Q70D, N128D or N128E and N303D or N303E are identified in A. ramosus peroxidase as sites for mutagenesis. Residues are substituted below and further analysis done:
  • the subset RESTx contains only four residues: S9, S334, G335 and P336, all of which are >5% solvent exposed.
  • the mutations S9D, S9E, S334D, S334E, G335D, G335E, P336D and P336E are proposed in A. ramosus peroxidase.
  • Acidic residues within the subset ACTSITE are: E44, D57, D77, E87, E176, D179, E190, D202, D209, D246 and the N-terminal carboxylic acid on P344. Of these only E44, D77, E176, D179, E190, D209, D246 and the N-terminal carboxylic acid on P344 are solvent exposed.
  • Suitable sites for mutations are E44Q, D77N, E176Q, D179N, E190Q, D209N and D246N.
  • D246N and D246E are risky mutations due to D246's importance for binding of heme.
  • the N-terminal 8 residues were not included in the calculations above, as they do not appear in the structure. None of these 8 residues, QGPGGGG, contain carboxylic groups. The following variants are proposed as possible mutations to enable attachment to this region: Q1E, Q1D, G2E, G2D, P3E, P3D, G4E, G4D, G5E, G5D, G6E, G6D, G7E, G7D, G8E, G8D.
  • mPEG 15,000 was suspended in toluene (4 ml/g of mPEG) 20% was distilled off at normal pressure to dry the reactants azeotropically.
  • Dichloromethane dry 1 ml/g mPEG
  • phosgene in toluene (1.93 M 5 mole/mole mPEG) was added and mixture stirred at room temperature overnight. The mixture was evaporated to dryness and the desired product was obtained as waxy lumps.
  • PD498 site-directed variants were constructed using the “maxi-oligonucleotide-PCR” method described by Sarkar et al., 1990, BioTechniques, 8, 404-407.
  • the template plasmid was shuttle vector pPD498 or an analogue of this containing a variant of the PD498 protease gene.
  • a PCR fragment of 769 bp was ligated into the pPD498 plasmid prepared by Bst E II and Bgl II digestion. Positive variants were recognized by Styl digestion and verified by DNA sequencing of the total 769 bp insert.
  • a PCR fragment of 769 bp was ligated into the pPD498 plasmid prepared by Bst E II and Bgl II digestion. Positive variants were recognized by ClaI digestion and verified by DNA sequencing of the total 769 bp insert.
  • a PCR fragment of 769 bp was ligated into the pPD498 plasmid prepared by Bst E II and Bgl II digestion. Positive variants were recognized by the absence of an Rsa I restriction site and verified by DNA sequencing of the total 769 bp insert.
  • synthetic oligonucleotides having the sequence GGG ATG TAA CCA AGG GAA GCA GCA CTC AAA CG (SEQ ID NO: 7) and the sequence CGA CTT TAT CGA TAA GGA CAA TAA CCC (SEQ ID NO: 8) were used simultaneously.
  • a PCR fragment of 769 bp was ligated into the pPD498 plasmid prepared by Bst E II and Bgl II digestion. Positive variants were recognized by Styl and ClaI digestion and verified by DNA sequencing of the total 769 bp insert.
  • synthetic oligonucleotides having the sequence GGG ATG TAA CCA AGG GAA GCA GCA CTC AAA CG (SEQ ID NO: 7), the sequence CGA CTT TAT CGA TAA GGA CAA TAA CCC (SEQ ID NO: 8) and the sequence CAA TGT ATC CAA AAC GTT CCA ACC AGC (SEQ ID NO: 9) were used simultaneously.
  • a PCR fragment of 769 bp was ligated into the pPD498 plasmid prepared by Bst E II and Bgl II digestion. Positive variants were recognized by Styl and ClaI digestion and absence of an Rsa I site. The variant was verified by DNA sequencing of the total 769 bp insert.
  • Vectors hosting the above mentioned PD498 variants were purified from E. coli cultures and transformed into B. subtilis in which organism the variants were fermented, expressed and purified as described in the “Materials and Methods” section above.
  • the molecule weight of the obtained derivative was approximately 120 kDa, corresponding to about 16 moles of mPEG attached per mole enzyme.
  • Dunkin Hartley guinea pigs are stimulated with 1.0 microgram PD498-SPEG 5,000 and 1.0 microgram modified variant PD498-SPEG 5,000 by intratracheal installation.
  • the IgG 1 levels of Dunkin Hartley guinea pigs during the trial period of 10 weeks are observed.
  • Humicola lanuginosa lipase (SEQ ID NO: 6) is available in Brookhaven Databank as ltib.pdb.
  • the lipase consists of 269 amino acids.
  • H. lanuginosa lipase The procedure described in Example 1 was followed.
  • the sequence of H. lanuginosa lipase is shown below in the table listing solvent accessibility data for H. lanuginosa lipase.
  • H. lanuginosa residue numbering is used (1-269), and the active site residues (functional site) are S146, S201 and H258.
  • the synonym TIB is used for H. lanuginosa lipase.
  • REST contains the arginines Arg133, Arg139, Arg160, Arg179 and Arg 209
  • SUB5B contains Arg118 and R125.
  • lanuginosa lipase A18K, G31K, T32K, N33K, G38K, A40K, D48K, T50K, E56K, D57K, S58K, G59K, V60K, G61K, D62K, T64K, L78K, N88K, G91K, N92K, L93K, S105K, G106K, V120K, P136K, G225K, L227K, V228K, P229K, P250K, F262K.
  • the Humicola lanuginosa lipase variant E87K + D254K was constructed, expressed and purified as described in WO 92/05249.
  • the lipase variant E87K + D254K-SPEG conjugate was prepared as described in Example 7, except that the enzyme is the Humicola lanuginosa lipase variant (E87K + D254K) described in Example 11 and the polymer is mPEG 15,000.
  • mice were immunized by subcutaneous injection of:
  • the amount of protein for each batch was measured by optical density measurements. Blood samples (200 microliters) were collected from the eyes one week after the immunization, but before the following immunization. Serum was obtained by blood clotting, and centrifugation.
  • the IgG 1 response was determined by use of the Balb/C mice IgG 1 ELISA method as described above.
  • the antibody titers elicited by the conjugate i.e. lipase-SPEG15,000 ranged between 960 and 1920, and were only 2 to 4 ⁇ lower than the antibody titer of 3840 that was elicited by unmodified HL82-LIPOLASE (figure to the left).

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Abstract

The present invention relates to polypeptide-polymer conjugates having added and/or removed one or more attachment groups for coupling polymeric molecules on the surface of the polypeptide structure, a method for preparing polypeptide-polymer conjugates of the invention, the use of said conjugated for reducing the immunogenicity and allergenicity and compositions comprising said conjugate.

Description

    CROSS-REFERENCE TO RELATED APPLICATIONS
  • This application is divisional of application Ser. No. 09/705,185 filed Nov. 2, 2000, which is a divisional of application Ser. No. 09/024,532 filed Feb. 17, 1998 which is a continuation of PCT/DK98/00046 filed Feb. 6, 1998, which claims priority under 35 U.S.C. 119 of Danish application no. 0135/97 filed Feb. 6, 1997.
    • BACKGROUND OF THE INVENTION
  • 1. Field of the Invention
  • The present invention relates to polypeptide-polymer conjugates having added and/or removed one or more attachment groups for coupling polymeric molecules on the surface of the 3D structure of the polypeptide, a method for preparing polypeptide-polymer conjugates of the invention, the use of said conjugated for reducing the immunogenicity and allergenicity, and compositions comprising said conjugate.
  • 2. Description of Related Art
  • The use of polypeptides, including enzymes, in the circulatory system to obtain a particular physiological effect is well-known in the medical arts. Further, within the arts of industrial applications, such as laundry washing, textile bleaching, person care, contact lens cleaning, food and feed preparation enzymes are used as a functional ingredient. One of the important differences between pharmaceutical and industrial application is that for the latter type of applications (i.e. industrial applications) the polypeptides (often enzymes) are not intended to enter into the circulatory system of the body.
  • Certain polypeptides and enzymes have an unsatisfactory stability and may under certain circumstances—dependent on the way of challenge—cause an immune response, typically an IgG and/or IgE response.
  • It is today generally recognized that the stability of polypeptides is improved and the immune response is reduced when polypeptides, such as enzymes, are coupled to polymeric molecules. It is believed that the reduced immune response is a result of the shielding of (the) epitope(s) on the surface of the polypeptide responsible for the immune response leading to antibody formation by the coupled polymeric molecules.
  • Techniques for conjugating polymeric molecules to polypeptides are well-known in the art.
  • One of the first commercially suitable techniques was described back in the early 1970's and disclosed in e.g. U.S. Pat. No. 4,179,337. Said patent concerns non-immunogenic polypeptides, such as enzymes and peptide hormones coupled to polyethylene glycol (PEG) or polypropylene glycol (PPG). At least 15% of polypeptides' physiological activity is maintained.
  • GB patent no. 1,183,257 (Crook et al.) describes chemistry for conjugation of enzymes to polysaccharides via a triazine ring.
  • Further, techniques for maintaining of the enzymatic activity of enzyme-polymer conjugates are also known in the art.
  • WO 93/15189 (Veronese et al.) concerns a method for maintaining the activity in polyethylene glycol-modified proteolytic enzymes by linking the proteolytic enzyme to a macromolecularized inhibitor. The conjugates are intended for medical applications.
  • It has been found that the attachment of polymeric molecules to a polypeptide often has the effect of reducing the activity of the polypeptide by interfering with the interaction between the polypeptide and its substrate. EP 183 503 (Beecham Group PLC) discloses a development of the above concept by providing conjugates comprising pharmaceutically useful proteins linked to at least one water-soluble polymer by means of a reversible linking group.
  • EP 471,125 (Kanebo) discloses skin care products comprising a parent protease (Bacillus protease with the trade name Esperase®) coupled to polysaccharides through a triazine ring to improve the thermal and preservation stability. The coupling technique used is also described in the above mentioned GB patent no. 1,183,257 (Crook et al.).
  • JP 3083908 describes a skin cosmetic material which contains a transglutaminase from guinea pig liver modified with one or more water-soluble substances such as PEG, starch, cellulose etc. The modification is performed by activating the polymeric molecules and coupling them to the enzyme. The composition is stated to be mild to the skin.
  • However, it is not always possible to readily couple polymeric molecules to polypeptides and enzymes. Further, there is still a need for polypeptide-polymer conjugates with an even more reduced immunogenicity and/or allergenicity.
    • SUMMARY OF THE INVENTION
  • It is the object of the present invention to provide improved polypeptide-polymer conjugates suitable for industrial and pharmaceutical applications.
  • The term “improved polypeptide-polymer conjugates” means in the context of the present invention conjugates having a reduced immune response in humans and animals and/or an improved stability. As will be described further below the immune response is dependent on the way of challenge.
  • The present inventors have found that polypeptides, such as enzymes, may be made less immunogenic and/or allergenic by adding and/or removing one or more attachment groups on the surface of the parent polypeptide to be coupled to polymeric molecules.
  • When introducing pharmaceutical polypeptide directly into the circulatory system (i.e. bloodstream) the potential risk is an immunogenic response in the form of mainly IgG, IgA and/or IgM antibodies. In contrast hereto, industrial polypeptides, such as enzymes used as a functional ingredient in e.g. detergents, are not intended to enter the circulatory system. The potential risk in connection with industrial polypeptides is inhalation causing an allergenic response in the form of mainly IgE antibody formation.
  • Therefore, in connection with industrial polypeptides the potential risk is respiratory allergenicity caused by inhalation, intratracheal and intranasal presentation of polypeptides.
  • The main potential risk of pharmaceutical polypeptides is immunogenicity caused by intradermal, intravenous or subcutaneous presentation of the polypeptide.
  • It is to be understood that reducing the “immunogenicity” and reducing the “respiratory allergenicity” are two very different problems based on different routes of exposure and on two very different immunological mechanisms:
  • The term “immunogenicity” used in connection with the present invention may be referred to as allergic contact dermatitis in a clinical setting and is a cell mediated delayed immune response to chemicals that contact and penetrate the skin. This cell mediated reaction is also termed delayed contact hypersensitivity (type IV reaction according to Gell and Combs classification of immune mechanisms in tissue damage).
  • The term “allergenicity” or “respiratory allergenicity” is an immediate anaphylactic reaction (type I antibody-mediated reaction according to Gell and Combs) following inhalation of e.g. polypeptides.
  • According to the present invention it is possible to provide polypeptides with a reduced immune response and/or improved stability, which has a substantially retained residual activity.
  • The allergic and the immunogenic response are in one term, at least in the context of the present invention called the “immune response”.
  • In the first aspect the invention relates to a polypeptide-polymer conjugate having
      • a) one or more additional polymeric molecules coupled to the polypeptide having been modified in a manner to increase the number of attachment groups on the surface of the polypeptide in comparison to the number of attachment groups available on the corresponding parent polypeptide, and/or
      • b) one or more fewer polymeric molecules coupled to the polypeptide having been modified in a manner to decrease the number of attachment groups at or close to the functional site(s) of the polypeptide in comparison to the number of attachment groups available on the corresponding parent polypeptide.
  • The term “parent polypeptide” refers to the polypeptide to be modified by coupling to polymeric molecules. The parent polypeptide may be a naturally-occurring (or wild-type) polypeptide or may be a variant thereof prepared by any suitable means. For instance, the parent polypeptide may be a variant of a naturally-occurring polypeptide which has been modified by substitution, deletion or truncation of one or more amino acid residues or by addition or insertion of one or more amino acid residues to the amino acid sequence of a naturally-occurring polypeptide.
  • A “suitable attachment group” means in the context of the present invention any amino acid residue group on the surface of the polypeptide capable of coupling to the polymeric molecule in question.
  • Preferred attachment groups are amino groups of lysine residues and the N-terminal amino group. Polymeric molecules may also be coupled to the carboxylic acid groups (—COOH) of amino acid residues in the polypeptide chain located on the surface. Carboxylic acid attachment groups may be the carboxylic acid group of aspartate or glutamate and the C-terminal COOH-group.
  • A “functional site” means any amino acid residues and/or cofactors which are known to be essential for the performance of the polypeptide, such as catalytic activity, e.g. the catalytic triad residues, histidine, aspartate and serine in serine proteases, or e.g. the heme group and the distal and proximal histidines in a peroxidase such as the Arthromyces ramosus peroxidase.
  • In the second aspect the invention relates to a method for preparing improved polypeptide-polymer conjugates comprising the steps of:
      • a) identifying amino acid residues located on the surface of the 3D structure of the parent polypeptide in question,
      • b) selecting target amino acid residues on the surface of said 3D structure of said parent polypeptide to be mutated,
      • c) i) substituting or inserting one or more amino acid residues selected in step b) with an amino acid residue having a suitable attachment group, and/or ii) substituting or deleting one or more amino acid residues selected in step b) at or close to the functional site(s),
      • d) coupling polymeric molecules to the mutated polypeptide.
  • The invention also relates to the use of a conjugate of the invention and the method of the invention for reducing the immunogenicity of pharmaceuticals and reducing the allergenicity of industrial products.
  • Finally the invention relates to compositions comprising a conjugate of the invention and further ingredients used in industrial products or pharmaceuticals.
    • BRIEF DESCRIPTION OF THE DRAWING
  • FIG. 1 shows the anti-lipase serum antibody levels after 5 weekly immunizations with i) control ii) unmodified lipase variant, iii) lipase variant-SPEG. (X: log(serum dilution); Y Optical Density (490/620)).
    • DETAILED DESCRIPTION OF THE INVENTION
  • It is the object of the present invention to provide improved polypeptide-polymer conjugates suitable for industrial and pharmaceutical applications.
  • Even though polypeptides used for pharmaceutical applications and industrial application can be quite different the principle of the present invention may be tailored to the specific type of parent polypeptide (i.e. enzyme, hormone peptides etc.).
  • The inventors of the present invention have provided improved polypeptide-polymer conjugates with a reduced immune response in comparison to conjugates prepared from the corresponding parent polypeptides.
  • The present inventors have found that polypeptides, such as enzymes, may be made less immunogenic and/or less allergenic by adding one or more attachment groups on the surface of the parent polypeptide. In addition thereto the inventors have found that a higher percentage of maintained residual functional activity may be obtained by removing attachment groups at or close to the functional site(s).
  • In the first aspect the invention relates to an improved polypeptide-polymer conjugate having
      • a) one or more additional polymeric molecules coupled to the polypeptide having been modified in a manner to increase the number of attachment groups on the surface of the polypeptide in comparison to the number of attachment groups available on the corresponding parent polypeptide, and/or
      • b) one or more fewer polymeric molecules coupled to the polypeptide having been modified in a manner to decrease the number of attachment groups at or close to the functional site(s) of the polypeptide in comparison to the number of attachment groups available on the corresponding parent polypeptide.
  • Whether the attachment groups should be added and/or removed depends on the specific parent polypeptide.
  • a) Addition of Attachment Groups
  • There may be a need for further attachment groups on the polypeptide if only few attachment groups are available on the surface of the parent polypeptide. The addition of one or more attachment groups by substituting or inserting one or more amino acid residues on the surface of the parent polypeptide increases the number of polymeric molecules which may be attached in comparison to the corresponding parent polypeptide. Conjugates with an increased number of polymeric molecules attached thereto are generally seen to have a reduced immune response in comparison to the corresponding conjugates having fewer polymeric molecules coupled thereto.
  • Any available amino acid residues on the surface of the polypeptide, preferentially not being at or close to the functional site(s), such as the active site(s) of enzymes, may in principle be subject to substitution and/or insertion to provide additional attachment groups.
  • As will be described further below the location of the additional coupled polymeric molecules may be of importance for the reduction of the immune response and the percentage of maintained residual functional activity of the polypeptide itself.
  • A conjugate of the invention may typically have from 1 to 25, preferentially 1 to 10 or more additional polymeric molecules coupled to the surface of the polypeptide in comparison to the number of polymeric molecules of a conjugate prepared on the basis of the corresponding parent polypeptide.
  • However, the optimal number of attachment groups to be added depends (at least partly) on the surface area (i.e. molecular weight) of the parent polypeptide to be shielded by the coupled polymeric molecules, and also on the number of already available attachment groups on the parent polypeptide.
  • b) Removing Attachment Groups
  • In the case of enzymes or other polypeptides performing their function by interaction with a substrate or the like, polymeric molecules coupled to the polypeptide might be impeded by the interaction between the polypeptide and its substrate or the like, if they are coupled at or close to the functional site(s) (i.e. active site of enzymes). This will most probably cause reduced activity.
  • In the case of enzymes having one or more polymeric molecules coupled at or close to the active site a substantial loss of residual enzymatic activity can be expected. Therefore, according to the invention conjugates may be constructed to maintain a higher percentage of residual enzymatic activity in comparison to a corresponding conjugates prepared on the basis of the parent enzyme in question. This may be done by substituting and/or deleting attachment groups at or close to the active site, hereby increasing the substrate affinity by improving the accessibility of the substrate in the catalytic cleft.
  • An enzyme-polymer conjugate of the invention may typically have from 1 to 25, preferably 1 to 10 fewer polymeric molecules coupled at or close to the active site in comparison to the number of polymeric molecules of a conjugate prepared on the basis of the corresponding parent polypeptide.
  • As will be explained below “at or close to” the functional site(s) means that no polymeric molecule(s) should be coupled within 5 Angstroms, preferably 8 Angstroms, especially 10 Angstroms of the functional site(s).
  • Removal of attachment groups at or close to the functional site(s) of the polypeptide may advantageously be combined with addition of attachment groups in other parts of the surface of the polypeptide.
  • The total number of attachment groups may this way be unchanged, increased or decreased. However the location(s) of the total number of attachment group(s) is(are) improved assessed by the reduction of the immune response and/or percentage of maintained residual activity. Improved stability may also be obtained this way.
  • The Number of Attachment Groups
  • Generally seen the number of attachment groups should be balanced to the molecular weight and/or surface area of the polypeptide. The more heavy the polypeptide is the more polymeric molecules should be coupled to the polypeptide to obtain sufficient shielding of the epitope(s) responsible for antibody formation.
  • Therefore, if the parent polypeptide molecule is relatively light (e.g. 1 to 35 kDa) it may be advantageous to increase the total number of coupled polymeric molecules (outside the functional site(s)) to a total between 4 and 20.
  • If the parent polypeptide molecules are heavier, for instance 35 to 60 kDa, the number of coupled polymeric molecules (outside the functional site(s)) may advantageously be increased to 7 to 40, and so on.
  • The ratio between the molecular weight (Mw) of the polypeptide in question and the number of coupled polymeric molecules considered to be suitable by the inventors is listed below in Table 1.
    TABLE 1
    Molecular weight of parent Number of polymeric molecules
    polypeptide (Mw) kDa coupled to the polypeptide
     1 to 35  4-20
    35 to 60  7-40
    60 to 80 10-50
    80 to 100 15-70
    More than 100 more than 20

    Reduced Immune Response vs. Maintained Residual Enzymatic Activity
  • Especially for enzymes, in comparison to many other types of polypeptides, there is a conflict between reducing the immune response and maintaining a substantial residual enzymatic activity as the activity of enzymes are connected with interaction between a substrate and the active site often present as a cleft in the enzyme structure.
  • Without being limited to any theory it is believed that the loss of enzymatic activity of enzyme-polymer conjugates might be a consequence of impeded access of the substrate to the active site in the form of spatial hindrance of the substrate by especially bulky and/or heavy polymeric molecules to the catalytic cleft. It might also, at least partly, be caused by disadvantageous minor structural changes of the 3D structure of the enzyme due to the stress made by the coupling of the polymeric molecules.
  • Maintained Residual Activity
  • A polypeptide-polymer conjugates of the invention has a substantially maintained functional activity.
  • A “substantially” maintained functional activity is in the context of the present invention defined as an activity which is at least between 20% and 30%, preferably between 30% and 40%, more preferably between 40% and 60%, better from 60% up to 80%, even better from 80% up to about 100%, in comparison to the activity of the conjugates prepared on the basis of corresponding parent polypeptides.
  • In the case of polypeptide-polymer conjugates of the invention where no polymeric molecules are coupled at or close to the functional site(s) the residual activity may even be up to 100% or very close thereto. If attachment group(s) of the parent polypeptide is(are) removed from the functional site the activity might even be more than 100% in comparison to modified (i.e. polymer coupled) parent polypeptide conjugate.
  • Position of Coupled Polymeric Molecules
  • To obtain an optimally reduced immune response (i.e. immunogenic and allergenic response) the polymeric molecules coupled to the surface of the polypeptide in question should be located in a suitable distance from each other.
  • In a preferred embodiment of the invention the parent polypeptide is modified in a manner whereby the polymeric molecules are spread broadly over the surface of the polypeptide. In the case of the polypeptide in question has enzymatic activity it is preferred to have as few as possible, especially none, polymeric molecules coupled at or close to the area of the active site.
  • In the present context “spread broadly over the surface of the polypeptide” means that the available attachment groups are located so that the polymeric molecules shield different parts of the surface, preferably the whole or close to the whole surface area away from the functional site(s), to make sure that epitope(s) are shielded and hereby not recognized by the immune system or its antibodies.
  • The area of antibody-polypeptide interaction typically covers an area of 500 Angstroms2, as described by Sheriff et al., 1987, Proc. Natl. Acad. Sci. USA, 84, 8075-8079. 500 Angstroms2 corresponds to a rectangular box of 25 Angstroms×20 Angstroms or a circular region of radius 12.6 Angstroms. Therefore, to prevent binding of antibodies to the epitope(s) to the polypeptide in question it is preferred to have a maximum distance between two attachment groups around 10 Angstroms.
  • Consequently, amino acid residues which are located in excess of 10 Angstroms away from already available attachment groups are suitable target residues. If two or more attachment groups on the polypeptide are located very close to each other it will in most cases result in that only one polymeric molecule will be coupled. To ensure a minimal loss of functional activity it is preferred not to couple polymeric molecules at or close to the functional site(s). Said distance depends at least partly on the bulkiness of the polymeric molecules to be coupled, as impeded access by the bulky polymeric molecules to the functional site is undesired. Therefore, the more bulky the polymeric molecules are the longer should the distance from the functional site to the coupled polymeric molecules be.
  • To maintain a substantial functional activity of the polypeptide in question attachment groups located within 5 Angstroms, preferred 8 Angstroms, especially 10 Angstroms from such functional site(s) should be left uncoupled and may therefore advantageously be removed or changed by mutation. Functional residues should normally not be mutated/removed, even though they potentially can be the target for coupling polymeric molecules. In said case it may thus be advantageous to choose a coupling chemistry involving different attachment groups.
  • Further, to provide a polypeptide having coupled polymeric molecules at (a) known epitope(s) recognizable by the immune system or close to said epitope(s) specific mutations at such sites are also considered advantageous according to the invention. If the position of the epitope(s) is(are) unknown it is advantageous to couple several or many polymeric molecules to the polypeptide.
  • As also mentioned above it is preferred that said attachment groups are spread broadly over the surface.
  • The Attachment Group
  • Virtually all ionized groups, such as the amino groups of lysine residues, are located on the surface of the polypeptide molecule (see for instance Thomas E. Creighton, 1993, “Proteins”, W.H. Freeman and Company, New York).
  • Therefore, the number of readily accessible attachment groups (e.g. amino groups) on a modified or parent polypeptide equals generally seen the number of lysine residues in the primary structure of the polypeptide plus the N-terminus amino group.
  • The chemistry of coupling polymeric molecules to amino groups are quite simple and well established in the art. Therefore, it is preferred to add and/or remove lysine residues (i.e. attachment groups) to/from the parent polypeptide in question to obtain improved conjugates with reduced immunogenicity and/or allergenicity and/or improved stability and/or high percentage maintained functional activity.
  • Polymeric molecules may also be coupled to the carboxylic groups (—COOH) of amino acid residues on the surface of the polypeptide. Therefore, if using carboxylic groups (including the C-terminal group) as attachment groups addition and/or removal of aspartate and glutamate residues may also be suitable according to the invention.
  • If using other attachment groups, such as —SH groups, they may be added and/or removed analogously.
  • Substitution of the amino acid residues is preferred over insertion, as the impact on the 3D structure of the polypeptide normally will be less pronounced.
  • Preferred substitutions are conservative substitutions. In the case of increasing the number of attachment groups the substitution may advantageously be performed at a location having a distance of 5 Angstroms, preferred 8 Angstroms, especially 10 Angstroms from the functional site(s) (active site for enzymes).
  • An example of a suitable conservative substitution to obtain an additional amino attachment group is an arginine to lysine substitution. Examples of conservative substitutions to obtain additional carboxylic attachment groups are aspargine to aspartate/glutamate or glutamine to aspartate/glutamate substitutions. To remove attachment groups a lysine residue may be substituted with an arginine and so on.
  • The Parent Polypeptide
  • In the context of the present invention the term “polypeptides” includes proteins, peptides and/or enzymes for pharmaceutical or industrial applications. Typically the polypeptides in question have a molecular weight in the range between about 1 to 100 kDa, often 15 kDa and 100 kDa.
  • Pharmaceutical Polypeptides
  • The term “pharmaceutical polypeptides” is defined as polypeptides, including peptides, such as peptide hormones, proteins and/or enzymes, being physiologically active when introduced into the circulatory system of the body of humans and/or animals.
  • Pharmaceutical polypeptides are potentially immunogenic as they are introduced into the circulatory system.
  • Examples of “pharmaceutical polypeptides” contemplated according to the invention include insulin, ACTH, glucagon, somatostatin, somatotropin, thymosin, parathyroid hormone, pigmentary hormones, somatomedin, erythropoietin, luteinizing hormone, chorionic gonadotropin, hypothalmic releasing factors, antidiuretic hormones, thyroid stimulating hormone, relaxin, interferon, thrombopoietin (TPO) and prolactin.
  • Industrial Polypeptides
  • Polypeptides used for industrial applications often have an enzymatic activity. Industrial polypeptides (e.g. enzymes) are (in contrast to pharmaceutical polypeptides) not intended to be introduced into the circulatory system of the body.
  • It is not very like that industrial polypeptides, such as enzymes used as ingredients in industrial compositions and/or products, such as detergents and personal care products, including cosmetics, come into direct contact with the circulatory system of the body of humans or animals, as such enzymes (or products comprising such enzymes) are not injected (or the like) into the bloodstream.
  • Therefore, in the case of the industrial polypeptide the potential risk is respiratory allergy (i.e. IgE response) as a consequence of inhalation to polypeptides through the respiratory passage.
  • In the context of the present invention “industrial polypeptides” are defined as polypeptides, including peptides, proteins and/or enzymes, which are not intended to be introduced into the circulatory system of the body of humans and/or animals.
  • Examples of such polypeptides are polypeptides, especially enzymes, used in products such as detergents, household article products, agrochemicals, personal care products, such as skin care products, including cosmetics and toiletries, oral and dermal pharmaceuticals, composition use for processing textiles, compositions for hard surface cleaning, and compositions used for manufacturing food and feed etc.
  • Enzymatic Activity
  • Pharmaceutical or industrial polypeptides exhibiting enzymatic activity will often belong to one of the following groups of enzymes including Oxidoreductases (E.C. 1, “Enzyme Nomenclature, (1992), Academic Press, Inc.), such as laccase and Superoxide dismutase (SOD); Transferases, (E.C. 2), such as transglutaminases (TGases); Hydrolases (E.C. 3), including proteases, especially subtilisins, and lipolytic enzymes; Isomerases (E.C. 5), such as Protein disulfide Isomerases (PDI).
  • Hydrolases
  • Proteolytic Enzymes
  • Contemplated proteolytic enzymes include proteases selected from the group of Aspartic proteases, such pepsins, cysteine proteases, such as papain, serine proteases, such as subtilisins, or metallo proteases, such as NEUTRASE®.
  • Specific examples of parent proteases include PD498 (WO 93/24623 and SEQ ID NO: 2), SAVINASE® (von der Osten et al., 1993, Journal of Biotechnology, 28, 55+, SEQ ID NO: 3), Proteinase K (Gunkel et al., 1989, Eur. J. Biochem, 179, 185-194), Proteinase R (Samal et al, 1990, Mol. Microbiol, 4, 1789-1792), Proteinase T (Samal et al., 1989, Gene, 85, p. 329-333), Subtilisin DY (Betzel et al. 1993, Arch. Biophys, 302(2), 499-502), Lion Y (JP 04197182-A), RENNILASE® (Available from Novo Nordisk A/S), JA16 (WO 92/17576), ALCALASE® (a natural subtilisin Carlberg variant) (von der Osten et al., 1993, Journal of Biotechnology, 28, 55+).
  • Lipolytic Enzymes
  • Contemplated lipolytic enzymes include Humicola lanuginosa lipases, e.g. the one described in EP 258 068 and EP 305 216 (See SEQ ID NO: 6 below), Humicola insolens, a Rhizomucor miehei lipase, e.g. as described in EP 238 023, Absidia sp. lipolytic enzymes (WO 96/13578), a Candida lipase, such as a C. antarctica lipase, e.g. the C. antarctica lipase A or B described in EP 214 761, a Pseudomonas lipase such as a P. alcaligenes and P. pseudoalcaligenes lipase, e.g. as described in EP 218 272, a P. cepacia lipase, e.g. as described in EP 331 376, a Pseudomonas sp. lipase as disclosed in WO 95/14783, a Bacillus lipase, e.g. a B. subtilis lipase (Dartois et al., 1993 Biochemica et Biophysica Acta 1131, 253-260), a B. stearothermophilus lipase (JP 64/744992) and a B. pumilus lipase (WO 91/16422). Other types of lipolytic enzymes include cutinases, e.g. derived from Pseudomonas mendocina as described in WO 88/09367, or a cutinase derived from Fusarium solani pisi (e.g. described in WO 90/09446).
  • Oxidoreductases
  • Laccases
  • Contemplated laccases include Polyporus pinisitus laccase (WO 96/00290), Myceliophthora laccase (WO 95/33836), Scytalidium laccase (WO 95/338337), and Pyricularia oryzae laccase (Available from Sigma).
  • Peroxidase
  • Contemplated peroxidases include B. pumilus peroxidases (WO 91/05858), Myxococcaceae peroxidase (WO 95/11964), Coprinus cinereus (WO 95/10602) and Arthromyces ramosus peroxidase (Kunishima et al. 1994, J. Mol. Biol., 235, 331-344).
  • Transferases
  • Transglutaminases
  • Suitable transferases include any transglutaminases disclosed in WO 96/06931 (Novo Nordisk A/S) and WO 96/22366 (Novo Nordisk A/S).
  • Isomerases
  • Protein Disulfide Isomerase
  • Without being limited thereto suitable protein disulfide isomerases include PDIs described in WO 95/01425 (Novo Nordisk A/S).
  • The Polymeric Molecule
  • The polymeric molecules coupled to the polypeptide may be any suitable polymeric molecule, including natural and synthetic homo-polymers, such as polyols (i.e. poly-OH), polyamines (i.e. poly-NH2) and polycarboxyl acids (i.e. poly-COOH), and further hetero-polymers i.e. polymers comprising one or more different coupling groups e.g. a hydroxyl group and amine groups.
  • Examples of suitable polymeric molecules include polymeric molecules selected from the group comprising polyalkylene oxides (PAO), such as polyalkylene glycols (PAG), including polyethylene glycols (PEG), methoxypolyethylene glycols (mPEG) and polypropylene glycols, PEG-glycidyl ethers (Epox-PEG), PEG-oxycarbonylimidazole (CDI-PEG), branched PEGs, polyvinyl alcohol (PVA), polycarboxylates, polyvinylpyrolidones, poly-D,L-amino acids, polyethylene-co-maleic acid anhydride, polystyrene-co-malic acid anhydride, dextrans including carboxymethyl-dextrans, heparin, homologous albumin, celluloses, including methylcellulose, carboxymethylcellulose, ethylcellulose, hydroxyethylcellulose carboxyethylcellulose and hydroxypropylcellulose, hydrolysates of chitosan, starches such as hydroxyethyl-starches and hydroxy propyl-starches, glycogen, agaroses and derivatives thereof, guar gum, pullulan, inulin, xanthan gum, carrageenin, pectin, alginic acid hydrolysates and bio-polymers.
  • Preferred polymeric molecules are non-toxic polymeric molecules such as (m)polyethylene glycol ((m)PEG) which further requires a relatively simple chemistry for its covalently coupling to attachment groups on the enzyme's surface.
  • Generally seen polyalkylene oxides (PAO), such as polyethylene oxides, such as PEG and especially mPEG, are the preferred polymeric molecules, as these polymeric molecules, in comparison to polysaccharides such as dextran, pullulan and the like, have few reactive groups capable of cross-linking.
  • Even though all of the above mentioned polymeric molecules may be used according to the invention the methoxypolyethylene glycols (mPEG) may advantageously be used. This arises from the fact that methoxyethylene glycols have only one reactive end capable of conjugating with the enzyme. Consequently, the risk of cross-linking is less pronounced. Further, it makes the product more homogeneous and the reaction of the polymeric molecules with the enzyme easier to control.
  • Preparation of Enzyme Variants
  • Enzyme variants to be conjugated may be constructed by any suitable method. A number of methods are well established in the art. For instance enzyme variants according to the invention may be generated using the same materials and methods described in e.g. WO 89/06279 (Novo Nordisk A/S), EP 130,756 (Genentech), EP 479,870 (Novo Nordisk A/S), EP 214,435 (Henkel), WO 87/04461 (Amgen), WO 87/05050 (Genex), EP application no. 87303761 (Genentech), EP 260,105 (Genencor), WO 88/06624 (Gist-Brocades NV), WO 88/07578 (Genentech), WO 88/08028 (Genex), WO 88/08033 (Amgen), WO 88/08164 (Genex), Thomas et al., 1985, Nature, 318, 375-376; Thomas et al., 1987, J. Mol. Biol., 193, 803-813; Russel and Fersht, 1987, Nature, 328, 496-500.
  • Generation of Site Directed Mutations
  • Prior to mutagenesis the gene encoding the polypeptide of interest must be cloned in a suitable vector. Methods for generating mutations in specific sites are described below.
  • Once the polypeptide encoding gene has been cloned, and desirable sites for mutation identified and the residue to substitute for the original ones have been decided, these mutations can be introduced using synthetic oligonucleotides. These oligonucleotides contain nucleotide sequences flanking the desired mutation sites; mutant nucleotides are inserted during oligo-nucleotide synthesis. In a preferred method, Site-directed mutagenesis is carried out by SOE-PCR mutagenesis technique described by Kammann et al., 1989, Nucleic Acids Research, 17(13), 5404, and by Sarkar G. and Sommer, S. S., 1990, Biotechniques, 8, 404-407.
  • Activation of Polymers
  • If the polymeric molecules to be conjugated with the polypeptide in question are not active it must be activated by the use of a suitable technique. It is also contemplated according to the invention to couple the polymeric molecules to the polypeptide through a linker. Suitable linkers are well-known to the skilled person.
  • Methods and chemistry for activation of polymeric molecules as well as for conjugation of polypeptides are intensively described in the literature. Commonly used methods for activation of insoluble polymers include activation of functional groups with cyanogen bromide, periodate, glutaraldehyde, biepoxides, epichlorohydrin, divinylsulfone, carbodiimide, sulfonyl halides, trichlorotriazine etc. (see R. F. Taylor, (1991), “Protein Immobilisation. Fundamentals and Applications”, Marcel Dekker, N.Y.; S. S. Wong, 1992, “Chemistry of Protein Conjugation and Crosslinking”, CRC Press, Boca Raton; G. T. Hermanson et al., 1993, “Immobilized Affinity Ligand Techniques”, Academic Press, N.Y.). Some of the methods concern activation of insoluble polymers but are also applicable to activation of soluble polymers e.g. periodate, trichlorotriazine, sulfonylhalides, divinylsulfone, carbodiimide etc. The functional groups being amino, hydroxyl, thiol, carboxyl, aldehyde or sulfydryl on the polymer and the chosen attachment group on the protein must be considered in choosing the activation and conjugation chemistry which normally consist of i) activation of polymer, ii) conjugation, and iii) blocking of residual active groups.
  • In the following a number of suitable polymer activation methods will be described shortly. However, it is to be understood that also other methods may be used.
  • Coupling polymeric molecules to the free acid groups of polypeptides may be performed with the aid of diimide and for example amino-PEG or hydrazino-PEG (Pollak et al., 1976, J. Amr. Chem. Soc., 98, 289-291) or diazoacetate/amide (Wong et al., 1992, “Chemistry of Protein Conjugation and Crosslinking”, CRC Press).
  • Coupling polymeric molecules to hydroxy groups are generally very difficult as it must be performed in water. Usually hydrolysis predominates over reaction with hydroxyl groups.
  • Coupling polymeric molecules to free sulfhydryl groups can be reached with special groups like maleimido or the ortho-pyridyl disulfide. Also vinylsulfone (U.S. Pat. No. 5,414,135, (1995), Snow et al.) has a preference for sulfhydryl groups but is not as selective as the other mentioned.
  • Accessible arginine residues in the polypeptide chain may be targeted by groups comprising two vicinal carbonyl groups.
  • Techniques involving coupling electrophilically activated PEGs to the amino groups of lysines may also be useful. Many of the usual leaving groups for alcohols give rise to an amine linkage. For instance, alkyl sulfonates, such as tresylates (Nilsson et al., 1984, Methods in Enzymology, 104, Jacoby, W. B., Ed., Academic Press: Orlando, 56-66; Nilsson et al., 1987, Methods in Enzymology, 135; Mosbach, K., Ed.; Academic Press: Orlando, 65-79; Scouten et al., 1987, Methods in Enzymology, 135, Mosbach, K., Ed., Academic Press: Orlando, 1987, 79-84; Crossland et al., 1971, J. Amr. Chem. Soc., 93, 4217-4219), mesylates (Harris, (1985), supra; Harris et al., 1984, J. Polym. Sci. Polym. Chem. Ed., 22, 341-352), aryl sulfonates like tosylates, and para-nitrobenzene sulfonates can be used.
  • Organic sulfonyl chlorides, e.g. tresyl chloride, effectively converts hydroxy groups in a number of polymers, e.g. PEG, into good leaving groups (sulfonates) that, when reacted with nucleophiles like amino groups in polypeptides allow stable linkages to be formed between polymer and polypeptide. In addition to high conjugation yields, the reaction conditions are in general mild (neutral or slightly alkaline pH, to avoid denaturation and little or no disruption of activity), and satisfy the non-destructive requirements to the polypeptide.
  • Tosylate is more reactive than the mesylate but also more unstable decomposing into PEG, dioxane, and sulfonic acid (Zalipsky, 1995, Bioconjugate Chem., 6, 150-165). Epoxides may also been used for creating amine bonds but are much less reactive than the above mentioned groups.
  • Converting PEG into a chloroformate with phosgene gives rise to carbamate linkages to lysines. This theme can be played in many variants substituting the chlorine with N-hydroxy succinimide (U.S. Pat. No. 5,122,614, (1992); Zalipsky et al., 1992, Biotechnol. Appl. Biochem., 15, 100-114; Monfardini et al., 1995, Bioconjugate Chem., 6, 62-69, with imidazole (Allen et al., 1991, Carbohydr. Res., 213, 309-319), with para-nitrophenol, DMAP (EP 632 082, 1993, Looze, Y.) etc. The derivatives are usually made by reacting the chloroformate with the desired leaving group. All these groups give rise to carbamate linkages to the peptide.
  • Furthermore, isocyanates and isothiocyanates may be employed yielding ureas and thioureas, respectively.
  • Amides may be obtained from PEG acids using the same leaving groups as mentioned above and cyclic imide thrones (U.S. Pat. No. 5,349,001 (1994), Greenwald et al.). The reactivity of these compounds is very high but may make the hydrolysis to fast.
  • PEG succinate made from reaction with succinic anhydride can also be used. The hereby comprised ester group make the conjugate much more susceptible to hydrolysis (U.S. Pat. No. 5,122,614, 1992, Zalipsky). This group may be activated with N-hydroxy succinimide.
  • Furthermore, a special linker can be introduced. The oldest being cyanuric chloride (Abuchowski et al., 1977, J. Biol. Chem., 252, 3578-3581; U.S. Pat. No. 4,179,337, 1979, Davis et al.; Shafer et al., 1986, J. Polym. Sci. Polym. Chem. Ed., 24, 375-378.
  • Coupling of PEG to an aromatic amine followed by diazotization yields a very reactive diazonium salt which in situ can be reacted with a peptide. An amide linkage may also be obtained by reacting an azlactone derivative of PEG (U.S. Pat. No. 5,321,095, 1994, Greenwald, R. B.) thus introducing an additional amide linkage.
  • As some peptides do not comprise many lysines, it may be advantageous to attach more than one PEG to the same lysine. This can be done e.g. by the use of 1,3-diamino-2-propanol.
  • PEGs may also be attached to the amino-groups of the enzyme with carbamate linkages (WO 95/11924, Greenwald et al.). Lysine residues may also be used as the backbone.
  • The coupling technique used in the examples is the N-succinimidyl carbonate conjugation technique described in WO 90/13590 (Enzon).
  • Method for Preparing Improved Conjugates
  • It is also an object of the invention to provide a method for preparing improved polypeptide-polymer conjugates comprising the steps of:
      • a) identifying amino acid residues located on the surface of the 3D structure of the parent polypeptide in question,
      • b) selecting target amino acid residues on the surface of said 3D structure of said parent polypeptide to be mutated,
      • c) i) substituting or inserting one or more amino acid residues selected in step b) with an amino acid residue having a suitable attachment group, and/or ii) substituting or deleting one or more amino acid residues selected in step b) at or close to the functional site(s),
      • d) coupling polymeric molecules to the mutated polypeptide.
        Step a) Identifying Amino Acid Residues Located on the Surface of the Parent Polypeptide
        3-Dimensional Structure (3D-Structure)
  • To perform the method of the invention a 3-dimensional structure of the parent polypeptide in question is required. This structure may for example be an X-ray structure, an NMR structure or a model-built structure. The Brookhaven Databank is a source of X-ray- and NMR-structures.
  • A model-built structure may be produced by the person skilled in the art if one or more 3D-structure(s) exist(s) of homologous polypeptide(s) sharing at least 30% sequence identity with the polypeptide in question. Several software packages exist which may be employed to construct a model structure. One example is the Homology 95.0 package from Biosym.
  • Typical actions required for the construction of a model structure are: alignment of homologous sequences for which 3D-structures exist, definition of Structurally Conserved Regions (SCRs), assignment of coordinates to SCRs, search for structural fragments/loops in structure databases to replace Variable Regions, assignment of coordinates to these regions, and structural refinement by energy minimization. Regions containing large inserts (>3 residues) relative to the known 3D-structures are known to be quite difficult to model, and structural predictions must be considered with care.
  • Having obtained the 3D-structure of the polypeptide in question, or a model of the structure based on homology to known structures, this structure serves as an essential prerequisite for the fulfillment of the method described below.
  • Step b) Selection of Target Amino Acid Residues for Mutation
  • Target amino acid residues to be mutated are according to the invention selected in order to obtain additional or fewer attachment groups, such as free amino groups (—NH2) or free carboxylic acid groups (—COOH), on the surface of the polypeptide and/or to obtain a more complete and broadly spread shielding of the epitope(s) on the surface of the polypeptide.
  • Conservative Substitution
  • It is preferred to make conservative substitutions in the polypeptide, as conservative substitutions secure that the impact of the mutation on the polypeptide structure is limited.
  • In the case of providing additional amino groups this may be done by substitution of arginine to lysine, which are both positively charged, but only the lysine having a free amino group suitable as an attachment group.
  • In the case of providing additional carboxylic acid groups the conservative substitution may for instance be an aspargine to aspartic acid or glutamine to glutamic acid substitution. These residues resemble each other in size and shape, except from the carboxylic groups being present on the acidic residues.
  • In the case of providing fewer attachment groups, e.g. at or close to the active site, a lysine may be substituted with an arginine, and so on.
  • Which amino acids to substitute depends in principle on the coupling chemistry to be applied.
  • Non-Conservative Substitution
  • The mutation may also be on target amino acid residues which are less/non-conservative. Such mutation is suitable for obtaining a more complete and broadly spread shielding of the polypeptide surface than can be obtained by the conservative substitutions.
  • The method of the invention is first described in general terms, and subsequently using specific examples.
  • Note the use of the following terms:
  • Attachment_residue: residue(s) which can bind polymeric molecules, e.g. lysines (amino group) or aspartic/glutamic acids (carboxylic groups). N- or C-terminal amino/carboxylic groups are to be included where relevant.
  • Mutation_residue: residue(s) which is to be mutated, e.g. arginine or aspargine/glutamine.
  • Essential_catalytic_residues: residues which are known to be essential for catalytic function, e.g. the catalytic triad in serine proteases.
  • Solvent_exposed_residues: These are defined as residues which are at least 5% exposed according to the BIOSYM/INSIGHT algorithm found in the module Homology 95.0. The sequence of commands is as follows: Homology=>ProStat=>Access_Surf=>Solv_Radius 1.4; Heavy atoms only; Radii source VdW; Output: Fractional Area; Polarity source: Default. The file filename_area.tab is produced. Note: For this program to function properly all water molecules must first be removed from the structure.
  • It looks for example like:
    # PD498FINALMODEL
    # residue area
    TRP_1 136.275711
    SER_2 88.188095
    PRO_3 15.458788
    ASN_4 95.322319
    ASP_5 4.903404
    PRO_6 68.096909
    TYR_7 93.333252
    TYR_8 31.791576
    SER_9 95.983139
    . . . continued
    • 1. Identification of residues which are more than 10 Angstroms away from the closest attachment_residue, and which are located at least 8 Angstroms away from essential_catalytic_residues. This residue subset is called REST, and is the primary region for conservative mutation_residue to attachment_residue substitutions.
    • 2. Identification of residues which are located in a 0-5 Angstroms shell around subset REST, but at least 8 Angstroms away from essential_catalytic_residues. This residue subset is called SUB5B. This is a secondary region for conservative mutation_residue to attachment_residue substitutions, as a ligand bound to an attachment_residue in SUB5B will extend into the REST region and potentially prevent epitope recognition.
    • 3. Identification of solvent_exposed mutation_residues in REST and SUB5B as potential mutation sites for introduction of attachment_residues.
    • 4. Use BIOSYM/INSIGHT's Biopolymer module and replace residues identified under action 3.
    • 5. Repeat 1-2 above producing the subset RESTx. This subset includes residues which are more than 10 Angstroms away from the nearest attachment_residue, and which are located at least 8 Angstroms away from essential catalytic residues.
    • 6. Identify solvent_exposed residues in RESTx. These are potential sites for less/non-conservative mutations to introduce atttachment_residues.
      Step c) Substituting, Inserting or Deleting Amino Acid Residues
  • The mutation(s) performed in step c) may be performed by standard techniques well known in the art, such as site-directed mutagenesis (see, e.g., Sambrook et al., 1989, Molecular Cloning. A Laboratory Manual, Cold Spring Harbor, N.Y.
  • A general description of nucleotide substitution can be found in e.g. Ford et al., 1991, Protein Expression and Purification, 2, 95-107.
  • Step d) Coupling Polymeric Molecules to the Modified Parent Enzyme
  • Polypeptide-polymer conjugates of the invention may be prepared by any coupling method known in the art including the above mentioned techniques.
  • Coupling of Polymeric Molecules to the Polypeptide in Question
  • If the polymeric molecules to be conjugated with the polypeptide are not active it must be activated by the use of a suitable method. The polymeric molecules may be coupled to the polypeptide through a linker. Suitable linkers are well known to the skilled person.
  • Methods and chemistry for activation of polymeric molecules as well as for conjugation of polypeptides are intensively described in the literature. Commonly used methods for activation of insoluble polymers include activation of functional groups with cyanogen bromide, periodate, glutaraldehyde, biepoxides, epichlorohydrin, divinylsulfone, carbodiimide, sulfonyl halides, trichlorotriazine etc. (see R. F. Taylor, 1991, “Protein Immobilisation. Fundamentals and Applications”, Marcel Dekker, N.Y.; S. S. Wong, 1992, “Chemistry of Protein Conjugation and Crosslinking”, CRC Press, Boca Raton; G. T. Hermanson et al., 1993, “Immobilized Affinity Ligand Techniques”, Academic Press, N.Y.). Some of the methods concern activation of insoluble polymers but are also applicable to activation of soluble polymers e.g. periodate, trichlorotriazine, sulfonylhalides, divinylsulfone, carbodiimide etc. The functional groups being amino, hydroxyl, thiol, carboxyl, aldehyde or sulfydryl on the polymer and the chosen attachment group on the protein must be considered in choosing the activation and conjugation chemistry which normally consists of i) activation of polymer, ii) conjugation, and iii) blocking of residual active groups.
  • In the following a number of suitable polymer activation methods will be described shortly. However, it is to be understood that also other methods may be used.
  • Coupling polymeric molecules to the free acid groups of enzymes can be performed with the aid of diimide and for example amino-PEG or hydrazino-PEG (Pollak et al., 1976, J. Amr. Chem. Soc., 98, 289-291) or diazoacetate/amide (Wong et al., 1992, “Chemistry of Protein Conjugation and Crosslinking”, CRC Press).
  • Coupling polymeric molecules to hydroxy groups are generally very difficult as it must be performed in water. Usually hydrolysis predominates over reaction with hydroxyl groups.
  • Coupling polymeric molecules to free sulfhydryl groups can be reached wih special groups like maleimido or the ortho-pyridyl disulfide. Also vinylsulfone (U.S. Pat. No. 5,414,135 (1995), Snow et al.) has a preference for sulfhydryl groups but is not as selective as the other mentioned.
  • Accessible arginine residues in the polypeptide chain may be targeted by groups comprising two vicinal carbonyl groups.
  • Techniques involving coupling electrophilically activated PEGs to the amino groups of lysines are also useful. Many of the usual leaving groups for alcohols give rise to an amine linkage. For instance, alkyl sulfonates, such as tresylates (Nilsson et al., 1984, Methods in Enzymology, 104, Jacoby, W. B., Ed., Academic Press: Orlando, 56-66; Nilsson et al., (1987), Methods in Enzymology, 135; Mosbach, K., Ed.; Academic Press: Orlando, 65-79; Scouten et al., 1987, Methods in Enzymology, 135, Mosbach, K., Ed., Academic Press: Orlando, 1987; 79-84; Crossland et al., 1971, J. Amr. Chem. Soc., 1971, 93, 4217-4219), mesylates (Harris, 1985, supra; Harris et al., 1984, J. Polym. Sci. Polym. Chem. Ed., 22, 341-352), aryl sulfonates like tosylates, and para-nitrobenzene sulfonates can be used.
  • Organic sulfonyl chlorides, e.g. tresyl chloride, effectively converts hydroxy groups in a number of polymers, e.g. PEG, into good leaving groups (sulfonates) that, when reacted with nucleophiles like amino groups in polypeptides allow stable linkages to be formed between polymer and polypeptide. In addition to high conjugation yields, the reaction conditions are in general mild (neutral or slightly alkaline pH, to avoid denaturation and little or no disruption of activity), and satisfy the non-destructive requirements to the polypeptide.
  • Tosylate is more reactive than the mesylate but also more unstable decomposing into PEG, dioxane, and sulfonic acid (Zalipsky, 1995, Bioconjugate Chem., 6, 150-165). Epoxides may also been used for creating amine bonds but are much less reactive than the above mentioned groups.
  • Converting PEG into a chloroformate with phosgene gives rise to carbamate linkages to lysines. This theme can be played in many variants substituting the chlorine with N-hydroxy succinimide (U.S. Pat. No. 5,122,614 (1992); Zalipsky et al., 1992, Biotechnol. Appl. Biochem., 15, 100-114; Monfardini et al., 1995, Bioconjugate Chem., 6, 62-69, with imidazole (Allen et al., 1991, Carbohydr. Res., 213, 309-319), with para-nitrophenol, DMAP (EP 632 082, 1993, Looze, Y.) etc. The derivatives are usually made by reacting the chloroformate with the desired leaving group. All these groups give rise to carbamate linkages to the peptide.
  • Furthermore, isocyanates and isothiocyanates may be employed yielding ureas and thioureas, respectively.
  • Amides may be obtained from PEG acids using the same leaving groups as mentioned above and cyclic imide thrones (U.S. Pat. No. 5,349,001 (1994), Greenwald et al.). The reactivity of these compounds is very high but may make the hydrolysis to fast.
  • PEG succinate made from reaction with succinic anhydride can also be used. The hereby comprised ester group makes the conjugate much more susceptible to hydrolysis (U.S. Pat. No. 5,122,614, (1992), Zalipsky). This group may be activated with N-hydroxy succinimide.
  • Furthermore, a special linker can be introduced. The oldest being cyanuric chloride (Abuchowski et al., 1977, J. Biol. Chem., 252, 3578-3581; U.S. Pat. No. 4,179,337, 1979, Davis et al.; Shafer et al., 1986, J. Polym. Sci. Polym. Chem. Ed., 24, 375-378).
  • Coupling of PEG to an aromatic amine followed by diazotization yields a very reactive diazonium salt which in situ can be reacted with a peptide. An amide linkage may also be obtained by reacting an azlactone derivative of PEG (U.S. Pat. No. 5,321,095, (1994), Greenwald, R. B.) thus introducing an additional amide linkage.
  • As some peptides do not comprise many lysines, it may be advantageous to attach more than one PEG to the same lysine. This can be done e.g. by the use of 1,3-diamino-2-propanol.
  • PEGs may also be attached to the amino-groups of the enzyme with carbamate linkages (WO 95/11924, Greenwald et al.). Lysine residues may also be used as the backbone.
  • Addition of Attachment Groups
  • Specific Examples of PD498 Variant-SPEG Conjugates
  • A specific example of a protease is the parent PD498 (WO 93/24623 and SEQ ID NO: 2). The parent PD498 has a molecular weight of 29 kDa.
  • Lysine and arginine residues are located as follows:
    Distance from the active site Arginine Lysine
     0-5 Angstroms 1
     5-10 Angstroms
    10-15 Angstroms 5 6
    15-20 Angstroms 2 3
    20-25 Angstroms 1 3
    Total 9 12
  • The inventors examined which parent PD498 sites on the surface may be suitable for introducing additional attachment groups.
  • A. Suitable conservative arginine to lysine substitutions in parent PD498 may be any of R51K, R62K, R121K, R169K, R250K, R28K, R190K.
  • B. Suitable non-conservative substitutions in parent PD498 may be any of P6K, Y7K, S9K, A10K, Y11K, Q12K, D43K, Y44K, N45K, N65K, G87K, 188K, N209K, A211K, N216K, N217K, G218K, Y219K, S220K, Y221K, G262K.
  • As there are no lysine residues at or close to the active site there is no need for removing any attachment group.
  • PD498 variant-SPEG conjugates may be prepared using any of the above mentioned PD498 variants as the starting material by any conjugation technique known in the art for coupling polymeric molecules to amino groups on the enzyme. A specific example is described below.
  • Removal of Attachment Groups
  • Specific Examples of BPN′ Variant-SPEG Conjugates
  • A specific example of a protease having an attachment group in the active site is BPN′ which has 11 attachment groups (plus an N-terminal amino group): BPN′ has a molecular weight of 28 kDa.
  • Lysine and arginine residues are located as follows:
    Distance from the active site Arginine Lysine
     0-5 Angstroms 1
     5-10 Angstroms
    10-15 Angstroms 1 4
    15-20 Angstroms 1 4
    20-25 Angstroms 2
    Total 2 11
  • The lysine residue located within 0-5 Angstroms of the active site can according to the invention advantageously be removed. Specifically this may be done by a K94R substitution.
  • BPN′ variant-SPEG conjugates may be prepared using the above mentioned BPN′ variant as the starting material by any conjugation technique known in the art for coupling polymeric molecules to amino groups on the enzyme.
  • Addition and Removal of Attachment Groups
  • Specific Example of SAVINASE®-SPEG Conjugates
  • As described in Example 2 parent SAVINASE® (von der Osten et al., 1993, Journal of Biotechnology, 28, 55+ and SEQ ID NO: 3) may according to the invention have added a number of amino attachment groups to the surface and removed an amino attachment group close to the active site.
  • Any of the following substitutions in SAVINASE® are sites for mutagenesis: R10K, R19K, R45K, R145K, R170K, R186K and R247K.
  • The substitution K94R is identified as a mutation suitable for preventing attachment of polymers close to active site.
  • SAVINASE® variant-SPEG conjugates may be prepared using any of the above mentioned SAVINASE® variants as the starting material by any conjugation technique known in the art for coupling polymeric molecules to amino groups on the enzyme.
  • Addition of Attachment Groups
  • Specific Examples of Humicola lanuginosa Lipase Variants-SPEG Conjugates
  • Specific examples of lipase variants with reduced immunogenicity using the parent Huminocal lanuginosa DSM 4109 lipase (see SEQ ID NO: 6) as the backbone for substitutions are listed below.
  • The parent unmodified Humicola lanuginosa lipase has 8 attachment groups including the N-terminal NH2 group and a molecular weight of about 29 kDa.
  • Suitable conservative arginine to lysine substitutions in the parent lipase may be any of R133K, R139K, R160K, R179K, R209K, R118K and R125K.
  • Suitable non-conservative substitutions in the parent lipase may be any of: A18K, G31K, T32K, N33K, G38K, A40K, D48K, T50K, E56K, D57K, S58K, G59K, V60K, G61K, D62K, T64K, L78K, N88K, G91K, N92K, L93K, S105K, G106K, V120K, P136K, G225K, L227K, V228K, P229K, P250K, F262K.
  • Further suitable non-conservative substitution in the Humicola lanuginosa lipase include: E87K or D254K.
  • Lipase variant-SPEG conjugates may be prepared using any of the above mentioned lipase variants as the starting material by any conjugation technique known in the art for coupling polymeric molecules to amino groups on the enzyme. A specific example is described below.
  • In Example 12 below it is shown that a conjugate of the Humicola lanuginosa lipase variant with E87K+ D254K substitutions coupled to S-PEG 15,000 has reduced immunogenic response in Balb/C mice in comparison to the corresponding parent unmodified enzyme.
  • Immunogenicity and Allergenicity
  • “Immunogenicity” is a broader term than “antigenicity” and “allergenicity”, and expresses the immune system's response to the presence of foreign substances. Said foreign substances are called immunogens, antigens and allergens depending of the type of immune response they elicit.
  • An “immunogen” may be defined as a substance which, when introduced into circulatory system of animals and humans, is capable of stimulating an immunologic response resulting in formation of immunoglobulin.
  • The term “antigen” refers to substances which by themselves are capable of generating antibodies when recognized as a non-self molecule.
  • Further, an “allergen” may be defined as an antigen which may give rise to allergic sensitization or an allergic response by IgE antibodies (in humans, and molecules with comparable effects in animals).
  • Assessment of Immunogenicity
  • Assessment of the immunogenicity may be made by injecting animal subcutaneously to enter the immunogen into the circulation system and comparing the response with the response of the corresponding parent polypeptide.
  • The “circulatory system” of the body of humans and animals means, in the context of the present invention, the system which mainly consists of the heart and blood vessels. The heart delivers the necessary energy for maintaining blood circulation in the vascular system. The circulation system functions as the organism's transportation system, when the blood transports O2, nutritious matter, hormones, and other substances of importance for the cell regulation into the tissue. Further the blood removes CO2 from the tissue to the lungs and residual substances to e.g. the kidneys. Furthermore, the blood is of importance for the temperature regulation and the defense mechanisms of the body, which include the immune system.
  • A number of in vitro animal models exist for assessment of the immunogenic potential of polypeptides. Some of these models give a suitable basis for hazard assessment in man. Suitable models include a mice model.
  • This model seeks to identify the immunogenic response in the form of the IgG response in Balb/C mice being injected subcutaneously with modified and unmodified polypeptides.
  • Also other animal models can be used for assessment of the immunogenic potential.
  • A polypeptide having “reduced immunogenicity” according to the invention indicates that the amount of produced antibodies, e.g. immunoglobulin in humans, and molecules with comparable effects in specific animals, which can lead to an immune response, is significantly decreased, when introduced into the circulatory system, in comparison to the corresponding parent polypeptide.
  • For Balb/C mice the IgG response gives a good indication of the immunigenic potential of polypeptides.
  • Assessment of Allergenicity
  • Assessment of allergenicity may be made by inhalation tests, comparing the effect of intratracheally (into the trachea) administrated parent enzymes with the corresponding modified enzymes according to the invention.
  • A number of in vivo animal models exist for assessment of the allegenicity of enzymes. Some of these models give a suitable basis for hazard assessment in man. Suitable models include a guinea pig model and a mouse model. These models seek to identify respiratory allergens as a function of elicitation reactions induced in previously sensitized animals. According to these models the alleged allergens are introduced intratracheally into the animals.
  • A suitable strain of guinea pigs, the Dunkin Hartley strain, does not as humans, produce IgE antibodies in connection with the allergic response. However, they produce another type of antibody the IgG1A and IgG1B (see e.g. Prentø, ATLA, 19, 8-14, 1991), which are responsible for their allergenic response to inhaled polypeptides including enzymes. Therefore, when using the Dunkin Hartley animal model, the relative amount of IgG1A and IgG1B is a measure of the allergenicity level.
  • The Balb/C mice strain is suitable for intratracheal exposure. Balb/C mice produce IgE as the allergic response.
  • More details on assessing respiratory allergens in guinea pigs and mice are described by Kimber et al., 1996, Fundamental and Applied Toxicology, 33, 1-10.
  • Other animals such as rats, rabbits etc. may also be used for comparable studies.
  • Composition
  • The invention relates to a composition comprising a polypeptide-polymer conjugate of the invention.
  • The composition may be a pharmaceutical or industrial composition.
  • The composition may further comprise other polypeptides, proteins or enzymes and/or ingredients normally used in e.g. detergents, including soap bars, household articles, agrochemicals, personal care products, including skin care compositions, cleaning compositions for e.g. contact lenses, oral and dermal pharmaceuticals, composition use for treating textiles, compositions used for manufacturing food, e.g. baking, and feed etc.
  • Use of the Polypeptide-Polymer Conjugate
  • The invention also relates to the use of the method of the invention for reducing the immune response of polypeptides.
  • It is also an object of the invention to use the polypeptide-polymer conjugate of the invention to reduce the allergenicity of industrial products, such as detergents, such as laundry, dish wash and hard surface cleaning detergents, and food or feed products.
  • Material and Methods
  • Materials
  • Enzymes:
    • PD498: Protease of subtilisin type shown in WO 93/24623. The sequence of PD498 is shown in SEQ ID NOS: 1 and 2.
    • SAVINASE® (Available from Novo Nordisk A/S)
    • Humicola lanuginosa lipase: Available from Novo Nordisk as LIPOLASE® and is further described in EP 305,216. The DNA and protein sequence is shown in SEQ ID NOS: 5 and 6, respectively.
      Strains:
  • B. subtilis 309 and 147 are variants of Bacillus lentus, deposited with the NCIB and accorded the accession numbers NCIB 10309 and 10147, and described in U.S. Pat. No. 3,723,250 incorporated by reference herein.
  • E. coli MC 1000 (M. J. Casadaban and S. N. Cohen (1980); J. Mol. Biol. 138 179-207), was made r−,m+ by conventional methods and is also described in US Patent Application Serial No. 039,298.
  • Vectors:
  • pPD498: E. coli-B. subtilis shuttle vector (described in U.S. Pat. No. 5,621,089 under section 6.2.1.6) containing the wild-type gene encoding for PD498 protease (SEQ ID NO: 2). The same vector is used for mutagenesis in E. coli as well as for expression in B. subtilis.
  • General Molecular Biology Methods:
  • Unless otherwise mentioned the DNA manipulations and transformations were performed using standard methods of molecular biology (Sambrook et al., 1989, Molecular cloning: A laboratory Manual, Cold Spring Harbor lab., Cold Spring Harbor, N.Y.; Ausubel, F. M. et al. (eds.) “Current protocols in Molecular Biology”. John Wiley and Sons, 1995; Harwood, C. R., and Cutting, S. M. (eds.) “Molecular Biological Methods for Bacillus”. John Wiley and Sons, 1990).
  • Enzymes for DNA manipulations were used according to the specifications of the suppliers.
  • Materials, Chemicals and Solutions:
  • Horse Radish Peroxidase labeled anti-rat-Ig (Dako, DK, P162, # 031; dilution 1:1000).
  • Mouse anti-rat IgE (Serotec MCA193; dilution 1:200).
  • Rat anti-mouse IgE (Serotec MCA419; dilution 1:100).
  • Biotin-labeled mouse anti-rat IgG1 monoclonal antibody (Zymed 03-9140; dilution 1:1000)
  • Biotin-labeled rat anti-mouse IgG1 monoclonal antibody (Serotec MCA336B; dilution 1:1000)
  • Streptavidin-horse radish peroxidase (Kirkegård & Perry 14-30-00; dilution 1:1000).
  • CovaLink NH2 plates (Nunc, Cat# 459439)
  • Cyanuric chloride (Aldrich)
  • Acetone (Merck)
  • Rat anti-Mouse IgG1, biotin (SeroTec, Cat# MCA336B)
  • Streptavidin, peroxidase (KPL)
  • Ortho-Phenylene-diamine (OPD) (Kem-en-Tec)
  • H2O2, 30% (Merck)
  • Tween 20 (Merck)
  • Skim Milk powder (Difco)
  • H2SO4 (Merck).
  • Buffers and Solutions:
    Carbonate buffer (0.1 M, pH 10 (1 liter)) Na2CO3 10.60 g
    PBS (pH 7.2 (1 liter)) NaCl  8.00 g
    KCl  0.20 g
    K2HPO4  1.04 g
    KH2PO4  0.32 g
    Washing buffer PBS, 0.05% (v/v) Tween 20
    Blocking buffer PBS, 2% (wt/v) Skim Milk powder
    Dilution buffer PBS, 0.05% (v/v) Tween 20, 0.5%
    (wt/v) Skim Milk powder
    Citrate buffer (0.1 M, pH 5.0-5.2 (1 liter)) NaCitrate 20.60 g
    Citric acid  6.30 g
  • Activation of CovaLink Plates:
  • Make a fresh stock solution of 10 mg cyanuric chloride per ml acetone.
  • Just before use, dilute the cyanuric chloride stock solution into PBS, while stirring, to a final concentration of 1 mg/ml.
  • Add 100 ml of the dilution to each well of the CovaLink NH2 plates, and incubate for 5 minutes at room temperature.
  • Wash 3 times with PBS.
  • Dry the freshly prepared activated plates at 50° C. for 30 minutes.
  • Immediately seal each plate with sealing tape.
  • Preactivated plates can be stored at room temperature for 3 weeks when kept in a plastic bag.
    • Sodium Borate, borax (Sigma)
    • 3,3-Dimethyl glutaric acid (Sigma)
    • CaCl2 (Sigma)
    • Tresyl chloride (2,2,2-triflouroethansulfonyl chloride) (Fluka)
    • 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (Fluka)
    • N-Hydroxy succinimide (Fluka art. 56480))
    • Phosgene (Fluka art. 79380)
    • Lactose (Merck 7656)
    • PMSF (phenyl methyl sulfonyl flouride) from Sigma
    • Succinyl-Alanine-Alanine-Proline-Phenylalanine-para-nitroanilide (Suc-AAPF-pNP) Sigma no. S-7388, Mw 624.6 g/mole.
      Coloring Substrate:
    • OPD: o-phenylene-diamine, (Kementec cat no. 4260).
      Test Animals:
    • Dunkin Hartley guinea pigs (from Charles River, DE)
    • Female Balb/C mice (about 20 grams) purchased from Bomholdtgaard, Ry, Denmark.
      Equipment:
    • XCEL II (Novex)
    • ELISA reader (UVmax, Molecular Devices)
    • HPLC (Waters)
    • PFLC (Pharmacia)
    • Superdex-75 column, Mono-Q, Mono S from Pharmacia, SW.
    • SLT: Fotometer from SLT LabInstruments
    • Size-exclusion chromatograph (Spherogel TSK-G2000 SW).
    • Size-exclusion chromatograph (Superdex 200, Pharmacia, SW)
    • Amicon Cell.
      Enzymes for DNA Manipulations
  • Unless otherwise mentioned all enzymes for DNA manipulations, such as e.g. restriction endonucleases, ligases etc., are obtained from New England Biolabs, Inc.
  • Methods
  • ELISA Procedure for Determination of IgG1 Positive Guinea Pigs
  • ELISA microtiter plates are coated with rabbit anti-PD498 1:8000 in carbonate buffer and incubated overnight at 4° C. The next day the plates are blocked with 2% BSA for 1 hour and washed 3 times with PBS Tween 20.
  • 1 microgram/ml PD498 is added to the plates and incubated for 1 hour, then washed 3 times with PBS Tween 20.
  • All guinea pig sera samples and controls are applied to the ELISA plates with 2 microliters sera and 98 microliters PBS, incubated for 1 hour and washed 3 times with PBS Tween 20.
  • Then goat anti-guinea pig IgG1 (1:4000 in PBS buffer (Nordic Immunology, 44-682)) is applied to the plates, incubated for 1 hour and washed with PBS tween 20.
  • Alkaline phosphatase marked rabbit anti-goat 1:8000 (Sigma A4187) is applied and incubated for 1 hour, washed 2 times in PBS Tween20 and 1 time with diethanol amine buffer.
  • The marked alkaline phosphatase is developed using p-nitrophenyl phosphate for 30 minutes at 37° C. or until appropriate color has developed.
  • The reaction is stopped using stop medium (K2HPO4/HaH3 buffer comprising EDTA (pH 10)) and read at OD 405/650 using an ELISA reader.
  • Double blinds are included on all ELISA plates.
  • Positive and negative sera values are calculated as the average blind values added 2 times the standard deviation. This gives an accuracy of 95%.
  • Determination of the Molecule Weight
  • Electrophoretic separation of proteins was performed by standard methods using 4-20% gradient SDS poly acrylamide gels (Novex). Proteins were detected by silver staining. The molecule weight was measured relative to the mobility of Mark-12@ wide range molecule weight standards from Novex.
  • Protease Activity
  • Analysis with Suc-Ala-Ala-Pro-Phe-pNa:
  • Proteases cleave the bond between the peptide and p-nitroaniline to give a visible yellow color absorbing at 405 nm.
  • Buffer: e.g. Britton and Robinson buffer pH 8.3.
  • Substrate: 100 mg suc-AAPF-pNa is dissolved into 1 ml dimethyl sulfoxide (DMSO). 100 microliters of this is diluted into 10 ml with Britton and Robinson buffer.
  • The substrate and protease solution is mixed and the absorbance is monitored at 405 nm as a function of time and ABS405 nm/min. The temperature should be controlled (20-50° C. depending on protease). This is a measure of the protease activity in the sample.
  • Proteolytic Activity
  • In the context of this invention proteolytic activity is expressed in Kilo NOVO Protease Units (KNPU). The activity is determined relatively to an enzyme standard (SAVINASE®), and the determination is based on the digestion of a dimethyl casein (DMC) solution by the proteolytic enzyme at standard conditions, i.e. 50° C., pH 8.3, 9 min. reaction time, 3 min. measuring time. A folder AF 220/1 is available upon request to Novo Nordisk A/S, Denmark, which folder is hereby included by reference.
  • A Glycine Unit (GU) is defined as the proteolytic enzyme activity which, under standard conditions, during a 15-minute incubation at 40° C., with N-acetyl casein as substrate, produces an amount of NH2-group equivalent to 1 mmole of glycine.
  • Enzyme activity can also be measured using the PNA assay, according to reaction with the soluble substrate succinyl-alanine-alanine-proline-phenyl-alanine-para-nitrophenol, which is described in Rothgeb, T. M., Goodlander, B. D., Garrison, P. H., and Smith, L. A., 1988 Journal of American Oil Chemists Society.
  • Fermentation of PD498 Variants
  • Fermentation of PD498 variants in B. subtilis are performed at 30° C. on a rotary shaking table (300 r.p.m.) in 500 ml baffled Erlenmeyer flasks containing 100 ml BPX medium for 5 days. In order to make an e.g. 2 liter broth 20 Erlenmeyer flasks are fermented simultaneously.
  • Media:
    BPX: Composition (per liter)
    Potato starch 100 g
    Ground barley 50 g
    Soybean flour 20 g
    Na2HPO4 × 12 H2O 9 g
    Pluronic 0.1 g
    Sodium caseinate 10 g
  • The starch in the medium is liquefied with alpha-amylase and the medium is sterilized by heating at 120° C. for 45 minutes. After sterilization the pH of the medium is adjusted to 9 by addition of NaHCO3 to 0.1 M.
  • Purification of PD498 Variants
  • Approximately 1.6 liters of PD498 variant fermentation broth are centrifuged at 5000 rpm for 35 minutes in 1 liter beakers. The supernatants are adjusted to pH 7.0 using 10% acetic acid and filtered on Seitz Supra S100 filter plates.
  • The filtrates are concentrated to approximately 400 ml using an Amicon CH2A UF unit equipped with an Amicon S1Y10 UF cartridge. The UF concentrate is centrifuged and filtered prior to absorption at room temperature on a Bacitracin affinity column at pH 7. The PD498 variant is eluted from the Bacitracin column at room temperature using 25% 2-propanol and 1 M sodium chloride in a buffer solution with 0.01 dimethylglutaric acid, 0.1 M boric acid and 0.002 M calcium chloride adjusted to pH 7.
  • The fractions with protease activity from the Bacitracin purification step are combined and applied to a 750 ml Sephadex G25 column (5 cm diameter) equilibrated with a buffer containing 0.01 dimethylglutaric acid, 0.1 M boric acid and 0.002 M calcium chloride adjusted to pH 6.0.
  • Fractions with proteolytic activity from the Sephadex G25 column are combined and applied to a 150 ml CM Sepharose CL 6B cation exchange column (5 cm diameter) equilibrated with a buffer containing 0.01 M dimethylglutaric acid, 0.1 M boric acid, and 0.002 M calcium chloride adjusted to pH 6.0.
  • The protease is eluted using a linear gradient of 0-0.5 M sodium chloride in 1 liter of the same buffer.
  • Protease containing fractions from the CM Sepharose column are combined and filtered through a 2 micron filter.
  • Balb/C Mice IgG ELISA Procedure:
  • The antigen is diluted to 1 mg/ml in carbonate buffer.
  • 100 ml is added to each well.
  • The plates are coated overnight at 4° C.
  • Unspecific adsorption is blocked by incubating each well for 1 hour at room temperature with 200 ml blocking buffer.
  • The plates are washed 3× with 300 ml washing buffer.
  • Unknown mouse sera are diluted in dilution buffer, typically 10×, 20× and 40×, or higher.
  • 100 ml is added to each well.
  • Incubation is for 1 hour at room temperature.
  • Unbound material is removed by washing 3× with washing buffer.
  • The anti-Mouse IgG1 antibody is diluted 2000× in dilution buffer.
  • 100 ml is added to each well.
  • Incubation is for 1 hour at room temperature.
  • Unbound material is removed by washing 3× with washing buffer.
  • Streptavidine is diluted 1000× in dilution buffer.
  • 100 ml is added to each well.
  • Incubation is for 1 hour at room temperature.
  • Unbound material is removed by washing 3× with 300 ml washing buffer.
  • OPD (0.6 mg/ml) and H2O2 (0.4 ml/ml) is dissolved in citrate buffer.
  • 100 ml is added to each well.
  • Incubation is for 10 minutes at room temperature.
  • The reaction is stopped by adding 100 ml H2SO4.
  • The plates are read at 492 nm with 620 nm as reference.
  • Immunization of Mice
  • Balb/C mice (20 grams) are immunized 10 times (intervals of 14 days) by subcutaneous injection of the modified or unmodified polypeptide in question, respectively by standard procedures known in art.
    • EXAMPLES Example 1
  • Suitable Substitutions in PD498 for Addition of Amino Attachment Groups (—NH2)
  • The 3D structure of parent PD498 was modeled as described above based on 59% sequence identity with Thermitase® (2tec.pdb).
  • The sequence of PD498 is SEQ ID NO: 2. PD498 residue numbering is used, 1-280.
  • The commands performed in Insight (BIOSYM) are shown in the command files makeKzone.bcl and makeKzone2.bcl below:
  • Conservative Substitutions:
    • makeKzone.bcl
    • 1 Delete Subset *
    • 2 Color Molecule Atoms * Specified Specification 55,0,255
    • 3 Zone Subset LYS :lys:NZ Static monomer/residue 10 Color_Subset 255,255,0
    • 4 Zone Subset NTERM :1:N Static monomer/residue 10 Color_Subset 255,255,0
    • 5 #NOTE: editnextline ACTSITE residues according to the protein
    • 6 Zone Subset ACTSITE :39,72,226 Static monomer/residue 8 Color_Subset 255,255,0
    • 7 Combine Subset ALLZONE Union LYS NTERM
    • 8 Combine Subset ALLZONE Union ALLZONE ACTSITE
    • 9 #NOTE: editnextline object name according to the protein
    • 10 Combine Subset REST Difference PD498FINALMODEL ALLZONE
    • 11 List Subset REST Atom Output_File restatom.list
    • 12 List Subset REST monomer/residue Output_File restmole.list
    • 13 Color Molecule Atoms ACTSITE Specified Specification 255,0,0
    • 14 List Subset ACTSITE Atom Output_File actsiteatom.list
    • 15 List Subset ACTSITE monomer/residue Output_File actsitemole.list
    • 16 #
    • 17 Zone Subset REST5A REST Static Monomer/Residue 5-Color_Subset
    • 18 Combine Subset SUB5A Difference REST5A ACTSITE
    • 19 Combine Subset SUB5B Difference SUB5A REST
    • 20 Color Molecule Atoms SUB5B Specified Specification 255,255,255
    • 21 List Subset SUB5B Atom Output_File sub5batom.list
    • 22 List Subset SUB5B monomer/residue Output_File sub5bmole.list
    • 23 #Now identify sites for lys->arg substitutions and continue with makezone2.bcl
    • 24 #Use grep command to identify ARG in restatom.list, sub5batom.list & accsiteatom.list.
      Comments:
  • Lines 1-8: The subset ALLZONE is defined as those residues which are either within 10 Angstroms of the free amino groups on lysines or the N-terminal, or within 8 Angstroms of the catalytic triad residues 39, 72 and 226.
  • Line 10: The subset REST is defined as those residues not included in ALLZONE.
  • Lines 17-20: Subset SUB5B is defined as those residues in a 5 Angstroms shell around REST, excluding residues within 8 Angstroms of the catalytic residues.
  • Line 23-24: REST contains Arg62 and Arg169, SUB5B contains Arg51, Arg121, and Arg250. ACTSITE contains Arg103, but position 103 is within 8 Angstroms from essential_catalytic_residues, and thus not relevant.
  • The color codes are: (255,0,255)=magenta, (255,255,0) yellow, (255,0,0) red, and (255, 255, 255)=white.
  • The substitutions R51K, R62K, R121K, R169K and R250K are identified in parent PD498 as suitable sites for mutagenesis. The residues are substituted below in section 2, and further analysis done:
  • Non-Conservative Substitutions:
    • makeKzone2.bcl
    • 1 #sourcefile makezone2.bcl Claus von der Osten 961128
    • 2 #
    • 3 #having scanned lists (grep arg command) and identified sites for lys->arg substitutions
    • 4 #NOTE: editnextline object name according to protein
    • 5 Copy Object -To_Clipboard -Displace PD498FINALMODEL newmodel 6 Biopolymer
    • 7 #NOTE: editnextline object name according to protein
    • 8 Blank Object On PD498FINALMODEL
    • 9 #NOTE: editnextlines with lys->arg positions Replace Residue newmodel:51 lys L
    • 11 Replace Residue newmodel:62 lys L
    • 12 Replace Residue newmodel:121 lys L
    • 13 Replace Residue newmodel:169 lys L
    • 14 Replace Residue newmodel:250 lys L
    • 15 #
    • 16 #Now repeat analysis done prior to arg->lys, now including introduced lysines
    • 17 Color Molecule Atoms newmodel Specified Specification 255,0,255
    • 18 Zone Subset LYSx newmodel:lys:NZ Static monomer/residue 10 Color_Subset 255,255,0
    • 19 Zone Subset NTERMx newmodel:l:N Static monomer/residue 10 Color_Subset 255,255,0
    • 20 #NOTE: editnextline ACTSITEx residues according to the protein
    • 21 Zone Subset ACTSITEx newmodel:39,72,226 Static monomer/residue 8 Color_Subset 255,255,0
    • 22 Combine Subset ALLZONEx Union LYSx NTERMx
    • 23 Combine Subset ALLZONEx Union ALLZONEx ACTSITEx
    • 24 Combine Subset RESTx Difference newmodel ALLZONEx List Subset RESTx Atom Output_File restxatom.list
    • 26 List Subset RESTx monomer/residue Output_File restxmole.list
    • 27 #
    • 28 Color Molecule Atoms ACTSITEx Specified Specification 255,0,0
    • 29 List Subset ACTSITEx Atom Output_File actsitexatom.list
    • 30 List Subset ACTSITEx monomer/residue Output_File actsitexmole.list
    • 31 #
    • 32 #read restxatom.list or restxmole.list to identify sites for (not_arg)->lys subst. if needed.
      Comments:
  • Lines 1-15: Solvent exposed arginines in subsets REST and SUB5B are replaced by lysines. Solvent accessibilities are recalculated following arginine replacement.
  • Lines 16-23: The subset ALLZONEx is defined as those residues which are either within 10 Angstroms of the free amino groups on lysines (after replacement) or the N-terminal, or within 8 Angstroms of the catalytic triad residues 39, 72 and 226.
  • Line 24-26: The subset RESTx is defined as those residues not included in ALLZONEx, i.e. residues which are still potential epitope contributors. Of the residues in RESTx, the following are >5% exposed (see lists below): 6-7,9-12, 43-45, 65, 87-88, 209, 211, 216-221, 262.
  • The following mutations are proposed in parent PD498: P6K, Y7K, S9K, A10K, Y11K, Q12K, D43K, Y44K, N45K, N65K, G87K, 188K, N209K, A211K, N216K, N217K, G218K, Y219K, S220K, Y221K, G262K.
  • Relevant Data for Example 1:
  • Solvent Accessibility Data for PD498MODEL:
    # PD498MODEL Fri Nov 29 10:24:48 MET 1996
    # residue area
    TRP_1 136.275711
    SER_2 88.188095
    PRO_3 15.458788
    ASN_4 95.322319
    ASP_5 4.903404
    PRO_6 68.096909
    TYR_7 93.333252
    TYR_8 31.791576
    SER_9 95.983139
    ALA_10 77.983536
    TYR_11 150.704727
    GLN_12 26.983349
    TYR_13 44.328232
    GLY_14 3.200084
    PRO_15 2.149547
    GLN_16 61.385445
    ASN_17 37.776707
    THR_18 1.237873
    SER_19 41.031750
    THR_20 4.321402
    PRO_21 16.658991
    ALA_22 42.107288
    ALA_23 0.000000
    TRP_24 3.713619
    ASP_25 82.645493
    VAL_26 74.397812
    THR_27 14.950654
    ARG_28 110.606209
    GLY_29 0.242063
    SER_30 57.225292
    SER_31 86.986198
    THR_32 1.928865
    GLN_33 42.008949
    THR_34 0.502189
    VAL_35 0.268693
    ALA_36 0.000000
    VAL_37 5.255383
    LEU_38 1.550332
    ASP_39 3.585718
    SER_40 2.475746
    GLY_41 4.329043
    VAL_42 1.704864
    ASP_43 25.889742
    TYR_44 89.194855
    ASN_45 109.981819
    HIS_46 0.268693
    PRO_47 66.580925
    ASP_48 0.000000
    LEU_49 0.770882
    ALA_50 49.618046
    ARG_51 218.751709
    LYS_52 18.808538
    VAL_53 39.937984
    ILE_54 98.478104
    LYS_55 103.612228
    GLY_56 17.199390
    TYR_57 67.719147
    ASP_58 0.000000
    PHE_59 40.291119
    ILE_60 50.151962
    ASP_61 70.078888
    ARG_62 166.777557
    ASP_63 35.892376
    ASN_64 120.641953
    ASN_65 64.982895
    PRO_66 6.986028
    MET_67 58.504269
    ASP_68 28.668840
    LEU_69 104.467468
    ASN_70 78.460953
    GLY_71 5.615932
    HIS_72 43.158905
    GLY_73 0.268693
    THR_74 0.000000
    HIS_75 0.484127
    VAL_76 1.880854
    ALA_77 0.000000
    GLY_78 0.933982
    THR_79 9.589676
    VAL_80 0.000000
    ALA_81 0.000000
    ALA_82 0.000000
    ASP_83 46.244987
    THR_84 27.783333
    ASN_85 75.924225
    ASN_86 44.813908
    GLY_87 50.453152
    ILE_88 74.428070
    GLY_89 4.115077
    VAL_90 6.717335
    ALA_91 2.872341
    GLY_92 0.233495
    MET_93 5.876057
    ALA_94 0.000000
    PRO_95 17.682203
    ASP_96 83.431740
    THR_97 1.506567
    LYS_98 72.674973
    ILE_99 4.251006
    LEU_100 6.717335
    ALA_101 0.806080
    VAL_102 1.426676
    ARG_103 2.662697
    VAL_104 2.171855
    LEU_105 18.808538
    ASP_106 52.167435
    ALA_107 52.905663
    ASN_108 115.871315
    GLY_109 30.943356
    SER_110 57.933651
    GLY_111 50.705326
    SER_112 56.383320
    LEU_113 71.312195
    ASP_114 110.410919
    SER_115 13.910152
    ILE_116 22.570246
    ALA_117 5.642561
    SER_118 29.313131
    GLY_119 0.000000
    ILE_120 1.343467
    ARG_121 118.391129
    TYR_122 44.203033
    ALA_123 0.000000
    ALA_124 7.974043
    ASP_125 83.851639
    GLN_126 64.311974
    GLY_127 36.812618
    ALA_128 4.705107
    LYS_129 90.886139
    VAL_130 1.039576
    LEU_131 2.149547
    ASN_132 4.315227
    LEU_133 1.880854
    SER_134 3.563334
    LEU_135 26.371397
    GLY_136 59.151070
    CYS_137 63.333755
    GLU_138 111.553314
    CYS_139 83.591461
    ASN_140 80.757843
    SER_141 25.899158
    THR_142 99.889725
    THR_143 73.323814
    LEU_144 5.589301
    LYS_145 94.708755
    SER_146 72.636993
    ALA_147 9.235920
    VAL_148 1.612160
    ASP_149 57.431465
    TYR_150 106.352493
    ALA_151 0.268693
    TRP_152 43.133667
    ASN_153 112.864975
    LYS_154 110.009468
    GLY_155 33.352180
    ALA_156 3.493014
    VAL_157 1.048144
    VAL_158 2.043953
    VAL_159 0.000000
    ALA_160 0.537387
    ALA_161 10.872165
    ALA_162 7.823834
    GLY_163 12.064573
    ASN_164 81.183388
    ASP_165 64.495300
    ASN_166 83.457443
    VAL_167 68.516815
    SER_168 78.799652
    ARG_169 116.937134
    THR_170 57.275074
    PHE_171 51.416462
    GLN_172 18.934589
    PRO_173 1.880854
    ALA_174 6.522357
    SER_175 26.184139
    TYR_176 21.425076
    PRO_177 85.613541
    ASN_178 34.700817
    ALA_179 0.268693
    ILE_180 1.074774
    ALA_181 3.761708
    VAL_182 0.000000
    GLY_183 2.149547
    ALA_184 0.951118
    ILE_185 0.806080
    ASP_186 30.022263
    SER_187 72.518509
    ASN_188 117.128021
    ASP_189 47.601345
    ARG_190 150.050873
    LYS_191 64.822807
    ALA_192 2.686934
    SER_193 96.223808
    PHE_194 51.482613
    SER_195 1.400973
    ASN_196 4.148808
    TYR_197 80.937309
    GLY_198 10.747736
    THR_199 93.221252
    TRP_200 169.943604
    VAL_201 15.280325
    ASP_202 12.141763
    VAL_203 0.268693
    THR_204 3.409728
    ALA_205 0.000000
    PRO_206 0.000000
    GLY_207 0.000000
    VAL_208 37.137192
    ASN_209 78.286270
    ILE_210 9.404268
    ALA_211 25.938599
    SER_212 5.037172
    THR_213 0.000000
    VAL_214 22.301552
    PRO_215 45.251030
    ASN_216 131.014160
    ASN_217 88.383461
    GLY_218 21.226780
    TYR_219 88.907570
    SER_220 39.966541
    TYR_221 166.037018
    MET_222 50.951096
    SER_223 54.435001
    GLY_224 1.880854
    THR_225 1.634468
    SER_226 17.432346
    MET_227 7.233279
    ALA_228 0.000000
    SER_229 0.000000
    PRO_230 0.268693
    HIS_231 2.680759
    VAL_232 0.000000
    ALA_233 0.000000
    GLY_234 1.074774
    LEU_235 11.500556
    ALA_236 0.000000
    ALA_237 0.000000
    LEU_238 1.612160
    LEU_239 0.000000
    ALA_240 10.648088
    SER_241 39.138004
    GLN_242 71.056175
    GLY_243 66.487144
    LYS_244 43.256012
    ASN_245 80.728127
    ASN_246 34.859673
    VAL_247 84.145645
    GLN_248 51.819775
    ILE_249 8.598188
    ARG_250 35.055809
    GLN_251 71.928093
    ALA_252 0.000000
    ILE_253 4.845899
    GLU_254 13.344438
    GLN_255 81.705254
    THR_256 9.836061
    ALA_257 2.810513
    ASP_258 44.656136
    LYS_259 113.071686
    ILE_260 32.089527
    SER_261 91.590103
    GLY_262 26.450439
    THR_263 38.308762
    GLY_264 46.870056
    THR_265 88.551804
    ASN_266 34.698349
    PHE_267 7.756911
    LYS_268 103.212852
    TYR_269 37.638382
    GLY_270 0.000000
    LYS_271 11.376978
    ILE_272 2.885231
    ASN_273 19.195255
    SER_274 2.651736
    ASN_275 38.177547
    LYS_276 84.549576
    ALA_277 1.074774
    VAL_278 4.775503
    ARG_279 162.693054
    TYR_280 96.572929
    CA_281 0.000000
    CA_282 0.000000
    CA_283 8.803203
    Subset REST:
    restmole.list
    Subset REST:
    PD498FINALMODEL: 6-7, 9-12, 43-46, 61-63, 65, 87-89, 111-114, 117-118, 131,
    PD498FINALMODEL: 137-139, 158-159, 169-171, 173-174, 180-181, 209, 211,
    PD498FINALMODEL: 216-221, 232-233, 262, E282H
    restatom.list
    Subset REST:
    PD498FINALMODEL: PRO 6: N, CA, CD, C, O, CB, CG
    PD498FINALMODEL: TYR 7: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ, OH
    PD498FINALMODEL: SER 9: N, CA, C, O, CB, OG
    PD498FINALMODEL: ALA 10: N, CA, C, O, CB
    PD498FINALMODEL: TYR 11: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ, OH
    PD498FINALMODEL: GLN 12: N, CA, C, O, CB, CG, CD, OE1, NE2
    PD498FINALMODEL: ASP 43: N, CA, C, O, CB, CG, OD1, OD2
    PD498FINALMODEL: TYR 44: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ, OH
    PD498FINALMODEL: ASN 45: N, CA, C, O, CB, CG, OD1, ND2
    PD498FINALMODEL: HIS 46: N, CA, C, O, CB, CG, ND1, CD2, CE1, NE2
    PD498FINALMODEL: ASP 61: N, CA, C, O, CB, CG, OD1, OD2
    PD498FINALMODEL: ARG 62: N, CA, C, O, CB, CG, CD, NE, CZ, NH1, NH2
    PD498FINALMODEL: ASP 63: N, CA, C, O, CB, CG, OD1, OD2
    PD498FINALMODEL: ASN 65: N, CA, C, O, CB, CG, OD1, ND2
    PD498FINALMODEL: GLY 87: N, CA, C, O
    PD498FINALMODEL: ILE 88: N, CA, C, O, CB, CG1, CG2, GD1
    PD498FINALMODEL: GLY 89: N, CA, C, O
    PD498FINALMODEL: GLY 111: N, CA, C, O
    PD498FINALMODEL: SER 112: N, CA, C, O, CB, OG
    PD498FINALMODEL: LEU 113: N, CA, C, O, CB, CG, CD1, CD2
    PD498FINALMODEL: ASP 114: N, CA, C, O, CB, CG, OD1, OD2
    PD498FINALMODEL: ALA 117: N, CA, C, O, CB
    PD498FINALMODEL: SER 118: N, CA, C, O, CB, OG
    PD498FINALMODEL: LEU 131: N, CA, C, O, CB, CG, CD1, CD2
    PD498FINALMODEL: CYS 137: N, CA, C, O, CB, SG
    PD498FINALMODEL: GLU 138: N, CA, C, O, CB, CG, CD, OE1, OE2
    PD498FINALMODEL: CYS 139: N, CA, C, O, CB, SG
    PD498FINALMODEL: VAL 158: N, CA, C, O, CB, CG1, CG2
    PD498FINALMODEL: VAL 159: N, CA, C, O, CB, CG1, CG2
    PD498FINALMODEL: ARG 169: N, CA, C, O, CB, CG, CD, NE, CZ, NH1, NH2
    PD498FINALMODEL: THR 170: N, CA, C, O, CB, OG1, CG2
    PD498FINALMODEL: PHE 171: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ
    PD498FINALMODEL: PRO 173: N, CA, CD, C, O, CB, CG
    PD498FINALMODEL: ALA 174: N, CA, C, O, CB
    PD498FINALMODEL: ILE 180: N, CA, C, O, CB, CG1, CG2, CD1
    PD498FINALMODEL: ALA 181: N, CA, C, O, CB
    PD498FINALMODEL: ASN 209: N, CA, C, O, CB, CG, OD1, ND2
    PD498FINALMODEL: ALA 211: N, CA, C, O, CB
    PD498FINALMODEL: ASN 216: N, CA, C, O, CB, CG, OD1, ND2
    PD498FINALMODEL: ASN 217: N, CA, C, O, CB, CG, OD1, ND2
    PD498FINALMODEL: GLY 218: N, CA, C, O
    PD498FINALMODEL: TYR 219: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ, OH
    PD498FINALMODEL: SER 220: N, CA, C, O, CB, OG
    PD498FINALMODEL: TYR 221: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ, OH
    PD498FINALMODEL: VAL 232: N, CA, C, O, CB, CG1, CG2
    PD498FINALMODEL: ALA 233: N, CA, C, O, CB
    PD498FINALMODEL: GLY 262: N, CA, C, O
    PD498FINALMODEL: CA E282H: CA
    Subset SUB5B:
    sub5bmole.list
    Subset SUB5B:
    PD498FINALMODEL: 4-5, 8, 13-16, 34-35, 47-51, 53, 64, 83, 85-86, 90-91,
    120-124,
    PD498FINALMODEL: 128-130, 140-141, 143-144, 147-148, 151-152, 156-157,
    PD498FINALMODEL: 165, 167-168, 172, 175-176, 178-179, 196, 200-205, 208,
    PD498FINALMODEL: 234-237, 250, 253-254, 260-261, 263-267, 272, E281H,
    PD498FINALMODEL: E283H
    sub5batom.list
    Subset SUB5B:
    PD498FINALMODEL: ASN 4: N, CA, C, O, CB, CG, OD1, ND2
    PD498FINALMODEL: ASP 5: N, CA, C, O, CB, CG, OD1, OD2
    PD498FINALMODEL: TYR 8: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ, OH
    PD498FINALMODEL: TYR 13: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ, OH
    PD498FINALMODEL: GLY 14: N, CA, C, O
    PD498FINALMODEL: PRO 15: N, CA, CD, C, O, CB, CG
    PD498FINALMODEL: GLN 16: N, CA, C, O, CB, CG, CD, OE1, NE2
    PD498FINALMODEL: THR 34: N, CA, C, O, CB, OG1, CG2
    PD498FINALMODEL: VAL 35: N, CA, C, O, CB, CG1, CG2
    PD498FINALMODEL: PRO 47: N, CA, CD, C, O, CB, CG
    PD498FINALMODEL: ASP 48: N, CA, C, O, CB, CG, OD1, OD2
    PD498FINALMODEL: LEU 49: N, CA, C, O, CB, CG, CD1, CD2
    PD498FINALMODEL: ALA 50: N, CA, C, O, CB
    PD498FINALMODEL: ARG 51: N, CA, C, O, CB, CG, CD, NE, CZ, NH1, NH2
    PD498FINALMODEL: VAL 53: N, CA, C, O, CB, CG1, CG2
    PD498FINALMODEL: ASN 64: N, CA, C, O, CB, CG, OD1, ND2
    PD498FINALMODEL: ASP 83: N, CA, C, O, CB, CG, OD1, OD2
    PD498FINALMODEL: ASN 85: N, CA, C, O, CB, CG, OD1, ND2
    PD498FINALMODEL: ASN 86: N, CA, C, O, CB, CG, OD1, ND2
    PD498FINALMODEL: VAL 90: N, CA, C, O, CB, CG1, CG2
    PD498FINALMODEL: ALA 91: N, CA, C, O, CB
    PD498FINALMODEL: ILE 120: N, CA, C, O, CB, CG1, CG2, CD1
    PD498FINALMODEL: ARG 121: N, CA, C, O, CB, CG, CD, NE, CZ, NH1, NH2
    PD498FINALMODEL: TYR 122: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ, OH
    PD498FINALMODEL: ALA 123: N, CA, C, O, CB
    PD498FINALMODEL: ALA 124: N, CA, C, O, CB
    PD498FINALMODEL: ALA 128: N, CA, C, O, CB
    PD498FINALMODEL: LYS 129: N, CA, C, O, CB, CG, CD, CE, NZ
    PD498FINALMODEL: VAL 130: N, CA, C, O, CB, CG1, CG2
    PD498FINALMODEL: ASN 140: N, CA, C, O, CB, CG, OD1, ND2
    PD498FINALMODEL: SER 141: N, CA, C, O, CB, OG
    PD498FINALMODEL: THR 143: N, CA, C, O, CB, OG1, CG2
    PD498FINALMODEL: LEU 144: N, CA, C, O, CB, CG, CD1, CD2
    PD498FINALMODEL: ALA 147: N, CA, C, O, CB
    PD498FINALMODEL: VAL 148: N, CA, C, O, CB, CG1, CG2
    PD498FINALMODEL: ALA 151: N, CA, C, O, CB
    PD498FINALMODEL: TRP 52: N, CA, C, O, CB, CG, CD1, CD2, NE1, CE2, CE3, CZ2,
    CZ3, CH2
    PD498FINALMODEL: ALA 156: N, CA, C, O, CB
    PD498FINALMODEL: VAL 157: N, CA, C, O, CB, CG1, CG2
    PD498FINALMODEL: ASP 165: N, CA, C, O, CB, CG, OD1, OD2
    PD498FINALMODEL: VAL 167: N, CA, C, O, CB, CG1, CG2
    PD498FINALMODEL: SER 168: N, CA, C, O, CB, OG
    PD498FINALMODEL: GLN 172: N, CA, C, O, CB, CG, CD, OE1, NE2
    PD498FINALMODEL: SER 175: N, CA, C, O, CB, OG
    PD498FINALMODEL: TYR 176: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ, OH
    PD498FINALMODEL: ASN 178: N, CA, C, O, CB, CG, OD1, ND2
    PD498FINALMODEL: ALA 179: N, CA, C, O, CB
    PD498FINALMODEL: ASN 196: N, CA, C, O, CB, CG, OD1, ND2
    PD498FINALMODEL: TRP 200: N, CA, C, O, CB, CG, CD1, CD2, NE1, CE2, CE3, CZ2,
    CZ3, CH2
    PD498FINALMODEL: VAL 201: N, CA, C, O, CB, CG1, CG2
    PD498FINALMODEL: ASP 202: N, CA, C, O, CB, CG, OD1, OD2
    PD498FINALMODEL: VAL 203: N, CA, C, O, CB, CG1, CG2
    PD498FINALMODEL: THR 204: N, CA, C, O, CB, OG1, CG2
    PD498FINALMODEL: ALA 205: N, CA, C, O, CB
    PD498FINALMODEL: VAL 208: N, CA, C, O, CB, CG1, CG2
    PD498FINALMODEL: GLY 234: N, CA, C, O
    PD498FINALMODEL: LEU 235: N, CA, C, O, CB, CG, CD1, CD2
    PD498FINALMODEL: ALA 236: N, CA, C, O, CB
    PD498FINALMODEL: ALA 237: N, CA, C, O, CB
    PD498FINALMODEL: ARG 250: N, CA, C, O, CB, CG, CD, NE, CZ, NH1, NH2
    PD498FINALMODEL: ILE 253: N, CA, C, O, CB, CG1, CG2, GD1
    PD498FINALMODEL: GLU 254: N, CA, C, O, CB, CG, CD, OE1, OE2
    PD498FINALMODEL: ILE 260: N, CA, C, O, CB, CG1, CG2, GD1
    PD498FINALMODEL: SER 261: N, CA, C, O, CB, OG
    PD498FINALMODEL: THR 263: N, CA, C, O, CB, OG1, CG2
    PD498FINALMODEL: GLY 264: N, CA, C, O
    PD498FINALMODEL: THR 265: N, CA, C, O, CB, OG1, CG2
    PD498FINALMODEL: ASN 266: N, CA, C, O, CB, CG, OD1, ND2
    PD498FINALMODEL: PHE 267: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ
    PD498FINALMODEL: ILE 272: N, CA, C, O, CB, CG1, CG2, CD1
    PD498FINALMODEL: CA E281H: CA
    PD498FINALMODEL: CA E283H: NA
    Subset ACTSITE:
    actsitemole.list
    Subset ACTSITE:
    PD498FINALMODEL: 36-42, 57-60, 66-80, 100-110, 115-116, 119, 132-136,
    160-164,
    PD498FINALMODEL: 182-184, 194, 206-207, 210, 212-215, 222-231
    actsiteatom.list
    Subset ACTSITE:
    PD498FINALMODEL: ALA 36: N, CA, C, O, CB
    PD498FINALMODEL: VAL 37: N, CA, C, O, CB, CG1, CG2
    PD498FINALMODEL: LEU 38: N, CA, C, O, CB, CG, CD1, CD2
    PD498FINALMODEL: ASP 39: N, CA, C, O, CB, CG, OD1, OD2
    PD498FINALMODEL: SER 40: N, CA, C, O, CB, OG
    PD498FINALMODEL: GLY 41: N, CA, C, O
    PD498FINALMODEL: VAL 42: N, CA, C, O, CB, CG1, CG2
    PD498FINALMODEL: TYR 57: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ, OH
    PD498FINALMODEL: ASP 58: N, CA, C, O, CB, CG, OD1, OD2
    PD498FINALMODEL: PHE 59: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ
    PD498FINALMODEL: ILE 60: N, CA, C, O, CB, CG1, CG2, CD1
    PD498FINALMODEL: PRO 66: N, CA, CD, C, O, CB, CG
    PD498FINALMODEL: MET 67: N, CA, C, O, CB, CG, SD, CE
    PD498FINALMODEL: ASP 68: N, CA, C, O, CB, CG, OD1, OD2
    PD498FINALMODEL: LEU 69: N, CA, C, O, CB, CG, CD1, CD2
    PD498FINALMODEL: ASN 70: N, CA, C, O, CB, CG, OD1, ND2
    PD498FINALMODEL: GLY 71: N, CA, C, O
    PD498FINALMODEL: HIS 72: N, CA, C, O, CB, CG, ND1, CD2, CE1, NE2
    PD498FINALMODEL: GLY 73: N, CA, C, O
    PD498FINALMODEL: THR 74: N, CA, C, O, CB, OG1, CG2
    PD498FINALMODEL: HIS 75: N, CA, C, O, CB, CG, ND1, CD2, CE1, NE2
    PD498FINALMODEL: VAL 76: N, CA, C, O, CB, CG1, CG2
    PD498FINALMODEL: ALA 77: N, CA, C, O, CB
    PD498FINALMODEL: GLY 78: N, CA, C, O
    PD498FINALMODEL: THR 79: N, CA, C, O, CB, OG1, CG2
    PD498FINALMODEL: VAL 80: N, CA, C, O, CB, CG1, CG2
    PD498FINALMODEL: LEU 100: N, CA, C, O, CB, CG, CD1, CD2
    PD498FINALMODEL: ALA 101: N, CA, C, O, CB
    PD498FINALMODEL: VAL 102: N, CA, C, O, CB, CG1, CG2
    PD498FINALMODEL: ARG 103: N, CA, C, O, CB, CG, CD, NE, CZ, NH1, NH2
    PD498FINALMODEL: VAL 104: N, CA, C, O, CB, CG1, CG2
    PD498FINALMODEL: LEU 105: N, CA, C, O, CB, CG, CD1, CD2
    PD498FINALMODEL: ASP 106: N, CA, C, O, CB, CG, OD1, OD2
    PD498FINALMODEL: ALA 107: N, CA, C, O, CB
    PD498FINALMODEL: ASN 108: N, CA, C, O, CB, CG, OD1, ND2
    PD498FINALMODEL: GLY 109: N, CA, C, O
    PD498FINALMODEL: SER 110: N, CA, C, O, CB, OG
    PD498FINALMODEL: SER 115: N, CA, C, O, CB, OG
    PD498FINALMODEL: ILE 116: N, CA, C, O, CB, CG1, CG2, CD1
    PD498FINALMODEL: GLY 119: N, CA, C, O
    PD498FINALMODEL: ASN 132: N, CA, C, O, CB, CG, OD1, ND2
    PD498FINALMODEL: LEU 133: N, CA, C, O, CB, CG, CD1, CD2
    PD498FINALMODEL: SER 134: N, CA, C, O, CB, OG
    PD498FINALMODEL: LEU 135: N, CA, C, O, CB, CG, CD1, CD2
    PD498FINALMODEL: GLY 136: N, CA, C, O
    PD498FINALMODEL: ALA 160: N, CA, C, O, CB
    PD498FINALMODEL: ALA 161: N, CA, C, O, CB
    PD498FINALMODEL: ALA 162: N, CA, C, O, CB
    PD498FINALMODEL: GLY 163: N, CA, C, O
    PD498FINALMODEL: ASN 164: N, CA, C, O, CB, CG, OD1, ND2
    PD498FINALMODEL: VAL 182: N, CA, C, O, CB, CG1, CG2
    PD498FINALMODEL: GLY 183: N, CA, C, O
    PD498FINALMODEL: ALA 184: N, CA, C, O, CB
    PD498FINALMODEL: PHE 194: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ
    PD498FINALMODEL: PRO 206: N, CA, CD, C, O, CB, CG
    PD498FINALMODEL: GLY 207: N, CA, C, O
    PD498FINALMODEL: ILE 210: N, CA, C, O, CB, CG1, CG2, CD1
    PD498FINALMODEL: SER 212: N, CA, C, O, CB, OG
    PD498FINALMODEL: THR 213: N, CA, C, O, CB, OG1, CG2
    PD498FINALMODEL: VAL 214: N, CA, C, O, CB, CG1, CG2
    PD498FINALMODEL: PRO 215: N, CA, CD, C, O, CB, CG
    PD498FINALMODEL: MET 222: N, CA, C, O, CB, CG, SD, CE
    PD498FINALMODEL: SER 223: N, CA, C, O, CB, OG
    PD498FINALMODEL: GLY 224: N, CA, C, O
    PD498FINALMODEL: THR 225: N, CA, C, O, CB, OG1, CG2
    PD498FINALMODEL: SER 226: N, CA, C, O, CB, OG
    PD498FINALMODEL: MET 227: N, CA, C, O, CB, CG, SD, CE
    PD498FINALMODEL: ALA 228: N, CA, C, O, CB
    PD498FINALMODEL: SER 229: N, CA, C, O, CB, OG
    PD498FINALMODEL: PRO 230: N, CA, CD, C, O, CB, CG
    PD498FINALMODEL: HIS 231: N, CA, C, O, CB, CG, ND1, CD2, CE1, NE2
    Subset RESTx:
    restxmole.list
    Subset RESTX:
    NEWMODEL: 6-7, 9-12, 43-46, 65, 87-89, 131, 173, 209, 211, 216-221, 232-233,
    NEWMODEL: 262, E282H
    restxatom.list
    Subset RESTX:
    NEWMODEL: PRO 6: N, CA, CD, C, O, CB, CG
    NEWMODEL: TYR 7: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ, OH
    NEWMODEL: SER 9: N, CA, C, O, CB, OG
    NEWMODEL: ALA 10: N, CA, C, O, CB
    NEWMODEL: TYR 11: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ, OH
    NEWMODEL: GLN 12: N, CA, C, O, CB, CG, CD, OE1, NE2
    NEWMODEL: ASP 43: N, CA, C, O, CB, CG, OD1, OD2
    NEWMODEL: TYR 44: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ, OH
    NEWMODEL: ASN 45: N, CA, C, O, CB, CG, OD1, ND2
    NEWMODEL: HIS 46: N, CA, C, O, CB, CG, ND1, CD2, CE1, NE2
    NEWMODEL: ASN 65: N, CA, C, O, CB, CG, OD1, ND2
    NEWMODEL: GLY 87: N, CA, C, O
    NEWMODEL: ILE 88: N, CA, C, O, CB, CG1, CG2, CD1
    NEWMODEL: GLY 89: N, CA, C, O
    NEWMODEL: LEU 131: N, CA, C, O, CB, CG, CD1, CD2
    NEWMODEL: PRO 173: N, CA, CD, C, O, CB, CG
    NEWMODEL: ASN 209: N, CA, C, O, CB, CG, OD1, ND2
    NEWMODEL: ALA 211: N, CA, C, O, CB
    NEWMODEL: ASN 216: N, CA, C, O, CB, CG, OD1, ND2
    NEWMODEL: ASN 217: N, CA, C, O, CB, CG, OD1, ND2
    NEWMODEL: GLY 218: N, CA, C, O
    NEWMODEL: TYR 219: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ, OH
    NEWMODEL: SER 220: N, CA, C, O, CB, OG
    NEWMODEL: TYR 221: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ, OH
    NEWMODEL: VAL 232: N, CA, C, O, CB, CG1, CG2
    NEWMODEL: ALA 233: N, CA, C, O, CB
    NEWMODEL: GLY 262: N, CA, C, O
    NEWMODEL: CA E282H: CA
    • Example 2
  • Suitable Substitutions in SAVINASE® for Addition of Amino Attachment Groups (—NH2)
  • The known X-ray structure of SAVINASE® was used to find where suitable amino attachment groups may is added (Betzel et al, 1992, J. Mol. Biol., 223, 427-445).
  • The 3D structure of SAVINASE® is available in the Brookhaven Databank as lsvn.pbd. A related subtilisin is available as 1st3.pdb.
  • The sequence of SAVINASE® is shown in SEQ ID NO: 3. The sequence numbering used is that of subtilisin BPN′, SAVINASE® having deletions relative to BPN′ at positions 36, 56, 158-159 and 163-164. The active site residues (functional site) are D32, H64 and S221.
  • The commands performed in Insight (BIOSYM) are shown in the command files makeKzone.bcl and makeKzone2.bcl below:
  • Conservative Substitutions:
    • makeKzone.bcl
    • Delete Subset *
    • Color Molecule Atoms * Specified Specification 255,0,255
    • Zone Subset LYS :lys:NZ Static monomer/residue 10 Color_Subset 255,255,0
    • Zone Subset NTERM :e1:N Static monomer/residue 10 Color_Subset 255,255,0
    • #NOTE: editnextline ACTSITE residues according to the protein
    • Zone Subset ACTSITE :e32,e64,e221 Static monomer/residue 8 Color_Subset 255,255,0
    • Combine Subset ALLZONE Union LYS NTERM
    • Combine Subset ALLZONE Union ALLZONE ACTSITE
    • #NOTE: editnextline object name according to the protein
    • Combine Subset REST Difference SAVI8 ALLZONE
    • List Subset REST Atom Output_File restatom.list
    • List Subset REST monomer/residue Output_File restmole.list
    • Color Molecule Atoms ACTSITE Specified Specification 255,0,0
    • List Subset ACTSITE Atom Output_File actsiteatom.list
    • List Subset ACTSITE monomer/residue Output_File actsitemole.list
    • #
    • Zone Subset REST5A REST Static Monomer/Residue 5-Color_Subset
    • Combine Subset SUB5A Difference REST5A ACTSITE
    • Combine Subset SUB5B Difference SUB5A REST
    • Color Molecule Atoms SUB5B Specified Specification 255,255,255
    • List Subset SUB5B Atom Output_File sub5batom.list
    • List Subset SUB5B monomer/residue Output_File subsbmole.list
    • #Now identify sites for lys->arg substitutions and continue with makezone2.bcl
    • #Use grep command to identify ARG in restatom.list, sub5batom.list & accsiteatom.list.
      Comments:
  • In this case of SAVINASE® REST contains the arginines Arg10, Arg170 and Arg 186, and SUB5B contains Arg19, Arg45, Arg145 and Arg247.
  • These residues are all solvent exposed. The substitutions R10K, R19K, R45K, R145K, R170K, R186K and R247K are identified in SAVINASE® as sites for mutagenesis within the scope of this invention. The residues are substituted below in section 2, and further analysis done. The subset ACTSITE contains Lys94.
  • The substitution K94R is a mutation removing lysine as attachment group close to the active site.
  • Non-Conservative Substitutions:
    • makeKzone2.bcl
    • #sourcefile makezone2.bcl Claus von der Osten 961128
    • #
    • #having scanned lists (grep arg command) and identified sites for lys->arg substitutions
    • #NOTE: editnextline object name according to protein
    • Copy Object -To_Clipboard -Displace SAVI8 newmodel
    • Biopolymer
    • #NOTE: editnextline object name according to protein
    • Blank Object On SAVI8
    • #NOTE: editnextlines with lys->arg positions
    • Replace Residue newmodel:e10 lys L
    • Replace Residue newmodel:e170 lys L
    • Replace Residue newmodel:e186 lys L
    • Replace Residue newmodel:e19 lys L
    • Replace Residue newmodel:e45 lys L
    • Replace Residue newmodel:e145 lys L
    • Replace Residue newmodel:e241 lys L
    • #
    • #Now repeat analysis done prior to arg->lys, now including introduced lysines
    • Color Molecule Atoms newmodel Specified Specification 255,0,255
    • Zone Subset LYSx newmodel:lys:NZ Static monomer/residue 10 Color_Subset 255,255,0
    • Zone Subset NTERMx newmodel:e1:N Static monomer/residue 10 Color_Subset 255,255,0
    • #NOTE: editnextline ACTSITEx residues according to the protein
    • Zone Subset ACTSITEx newmodel:e32,e64,e221 Static monomer/residue 8 Color_Subset 255,255,0
    • Combine Subset ALLZONEx Union LYSx NTERMx
    • Combine Subset ALLZONEx Union ALLZONEx ACTSITEx
    • Combine Subset RESTx Difference newmodel ALLZONEx
    • List Subset RESTx Atom Output_File restxatom.list
    • List Subset RESTx monomer/residue Output_File restxmole.list
    • #
    • Color Molecule Atoms ACTSITEx Specified Specification 255,0,0
    • List Subset ACTSITEx Atom Output_File actsitexatom.list
    • List Subset ACTSITEx monomer/residue Output_File actsitexmole.list
    • #
    • #read restxatom.list or restxmole.list to identify sites for (not_arg)->lys subst. if needed.
      Comments:
  • Of the residues in RESTx, the following are >5% exposed (see lists below): 5, 14, 22, 38-40, 42, 75-76, 82, 86, 103-105, 108, 133-135, 137, 140, 173, 204, 206, 211-213, 215-216, 269. The following mutations are proposed in SAVINASE®: P5K, P14K, T22K, T38K, H39K, P40K, L42K, L75K, N76K, L82K, P86K, S103K, V104K, S105K, A108K, A133K, T134K, L135K, Q137K, N140K, N173K, N204K, Q206K, G211K, S212K, T213K, A215K, S216K, N269K.
  • Relevant Data for Example 2:
  • Solvent Accessibility Data for SAVINASE®:
    # SAVI8NOH2O Fri Nov 29 13: 32: 07 MET 1996
    # residue area
    ALA_1 118.362808
    GLN_2 49.422764
    SER_3 61.982887
    VAL_4 71.620255
    PRO_5 21.737535
    TRP_6 58.718731
    GLY_7 4.328117
    ILE_8 6.664074
    SER_9 60.175900
    ARG_10 70.928963
    VAL_11 2.686934
    GLN_12 72.839996
    ALA_13 0.000000
    PRO_14 52.308453
    ALA_15 38.300892
    ALA_16 0.000000
    HIS_17 41.826324
    ASN_18 136.376602
    ARG_19 105.678642
    GLY_20 48.231510
    LEU_21 17.196377
    THR_22 36.781742
    GLY_23 0.000000
    SER_24 64.151276
    GLY_25 50.269905
    VAL_26 4.030401
    LYS_27 54.239555
    VAL_28 0.000000
    ALA_29 0.000000
    VAL_30 3.572827
    LEU_31 0.233495
    ASP_32 1.074774
    THR_33 1.973557
    GLY_34 3.638052
    ILE_35 8.044439
    SER_36 8.514903
    THR_37 122.598907
    HIS_38 18.834011
    PRO_39 76.570526
    ASP_40 0.000000
    LEU_41 19.684013
    ASN_42 88.870216
    ILE_43 56.117710
    ARG_44 110.647194
    GLY_45 26.935413
    GLY_46 35.515778
    ALA_47 21.495472
    SER_48 34.876190
    PHE_49 52.647541
    VAL_50 23.364208
    PRO_51 110.408752
    GLY_52 80.282906
    GLU_53 43.033707
    PRO_54 124.444336
    SER_55 60.284889
    THR_56 47.103241
    GLN_57 120.803505
    ASP_58 12.784743
    GLY_59 61.742443
    ASN_60 56.760231
    GLY_61 1.576962
    HIS_62 38.590118
    GLY_63 0.000000
    THR_64 0.537387
    HIS_65 0.968253
    VAL_66 1.612160
    ALA_67 0.000000
    GLY_68 2.801945
    THR_69 9.074596
    ILE_70 0.000000
    ALA_71 4.577205
    ALA_72 0.000000
    LEU_73 47.290039
    ASN_74 102.187248
    ASN_75 60.210400
    SER_76 84.614494
    ILE_77 66.098572
    GLY_78 17.979534
    VAL_79 5.642561
    LEU_80 13.025185
    GLY_81 0.000000
    VAL_82 0.268693
    ALA_83 0.000000
    PRO_84 18.193810
    SER_85 56.839039
    ALA_86 13.075745
    GLU_87 37.011765
    LEU_88 2.149547
    TYR_89 30.633518
    ALA_90 1.343467
    VAL_91 0.779450
    LYS_92 5.862781
    VAL_93 0.466991
    LEU_94 10.747736
    GLY_95 8.707102
    ALA_96 41.414677
    SER_97 96.066040
    GLY_98 33.374485
    SER_99 67.664116
    GLY_100 35.571117
    SER_101 54.096992
    VAL_102 52.695324
    SER_103 62.929684
    SER_104 8.683097
    ILE_105 15.852910
    ALA_106 14.509443
    GLN_107 94.463066
    GLY_108 0.000000
    LEU_109 0.537387
    GLU_110 63.227707
    TRP_111 55.500740
    ALA_112 0.502189
    GLY_113 11.908267
    ASN_114 107.208527
    ASN_115 78.811234
    GLY_116 41.453194
    MET_117 9.634291
    HIS_118 54.022118
    VAL_119 5.105174
    ALA_120 0.268693
    ASN_121 0.233495
    LEU_122 0.537387
    SER_123 4.004620
    LEU_124 21.927265
    GLY_125 55.952454
    SER_126 40.241180
    PRO_127 107.409439
    SER_128 57.988609
    PRO_129 85.021118
    SER_130 20.460915
    ALA_131 57.404362
    THR_132 74.438805
    LEU_133 12.091203
    GLU_134 73.382019
    GLN_135 114.870010
    ALA_136 2.122917
    VAL_137 1.074774
    ASN_138 55.622704
    SER_139 29.174965
    ALA_140 0.268693
    THR_141 27.962946
    SER_142 87.263145
    ARG_143 88.201218
    GLY_144 38.477882
    VAL_145 2.079151
    LEU_146 13.703363
    VAL_147 2.690253
    VAL_148 1.074774
    ALA_149 0.000000
    ALA_150 4.356600
    SER_151 0.000000
    GLY_152 12.628590
    ASN_153 84.248703
    SER_154 77.662354
    GLY_155 25.409861
    ALA_156 38.074570
    GLY_157 40.493744
    SER_158 53.915291
    ILE_159 4.352278
    SER_160 12.458543
    TYR_161 29.670284
    PRO_162 4.030401
    ALA_163 0.968253
    ARG_164 84.059120
    TYR_165 28.641129
    ALA_166 68.193314
    ASN_167 61.686481
    ALA_168 0.537387
    MET_169 0.586837
    ALA_170 0.000000
    VAL_171 0.000000
    GLY_172 0.000000
    ALA_173 0.933982
    THR_174 3.013133
    ASP_175 34.551376
    GLN_176 96.873039
    ASN_177 98.664368
    ASN_178 41.197159
    ASN_179 60.263512
    ARG_180 64.416336
    ALA_181 7.254722
    SER_182 91.590881
    PHE_183 52.126518
    SER_184 2.101459
    GLN_185 15.736279
    TYR_186 44.287792
    GLY_187 5.114592
    ALA_188 69.406563
    GLY_189 36.926083
    LEU_190 16.511177
    ASP_191 7.705349
    ILE_192 0.268693
    VAL_193 4.299094
    ALA_194 0.000000
    PRO_195 0.806080
    GLY_196 0.000000
    VAL_197 25.257177
    ASN_198 82.177422
    VAL_199 10.747736
    GLN_200 80.374527
    SER_201 2.008755
    THR_202 0.000000
    TYR_203 80.679886
    PRO_204 34.632195
    GLY_205 74.536827
    SER_206 74.964920
    THR_207 57.070065
    TYR_208 82.895500
    ALA_209 22.838940
    SER_210 69.045639
    LEU_211 49.708279
    ASN_212 86.905457
    GLY_213 2.686934
    THR_214 4.669909
    SER_215 15.225292
    MET_216 7.261287
    ALA_217 0.000000
    THR_218 0.000000
    PRO_219 0.806080
    HIS_220 2.662697
    VAL_221 0.268693
    ALA_222 0.000000
    GLY_223 0.000000
    ALA_224 7.206634
    ALA_225 1.039576
    ALA_226 0.268693
    LEU_227 1.074774
    VAL_228 1.541764
    LYS_229 39.262505
    GLN_230 54.501614
    LYS_231 81.154129
    ASN_232 30.004124
    PRO_233 91.917931
    SER_234 102.856705
    TRP_235 64.639481
    SER_236 51.797619
    ASN_237 24.866917
    VAL_238 78.458466
    GLN_239 73.981461
    ILE_240 14.474245
    ARG_241 41.242931
    ASN_242 64.644814
    HIS_243 50.671440
    LEU_244 5.127482
    LYS_245 48.820000
    ASN_246 115.264534
    THR_247 22.205376
    ALA_248 16.415077
    THR_249 60.503101
    SER_250 74.511597
    LEU_251 48.861599
    GLY_252 39.124340
    SER_253 49.811481
    THR_254 88.421982
    ASN_255 72.490181
    LEU_256 54.835758
    TYR_257 38.798912
    GLY_258 3.620916
    SER_259 35.017368
    GLY_260 0.537387
    LEU_261 8.598188
    VAL_262 4.519700
    ASN_263 16.763659
    ALA_264 3.413124
    GLU_265 37.942276
    ALA_266 15.871746
    ALA_267 3.947115
    THR_268 2.475746
    ARG_269 176.743362
    ION_270 0.000000
    ION_271 5.197493
    Subset REST:
    restmole.list
    Subset REST:
    SAVI8: E5-E15, E17-E18, E22, E38-E40, E42-E43, E73-E76, E82-E86,
    E103-E105,
    SAVI8: E108-E109, E111-E112, E115-E116, E122, E128-E144, E149-E150,
    E156-E157,
    SAVI8: E160-E162, E165-E168, E170-E171, E173, E180-E188, E190-E192, E200,
    SAVI8: E203-E204, E206, E211-E213, E215-E216, E227-E230, E255-E259,
    E261-E262,
    SAVI8: E267-E269
    restatom.list
    Subset REST:
    SAVI8: PRO E5: N, CD, CA, CG, CB, C, O
    SAVI8: TRP E6: N, CA, CD2, CE2, NE1, CD1, CG, CE3, CZ3, CH2, CZ2, CB, C, O
    SAVI8: GLY E7: N, CA, C, O
    SAVI8: ILE E8: N, CA, CD1, CG1, CB, CG2, C, O
    SAVI8: SER E9: N, CA, OG, CB, C, O
    SAVI8: ARG E10: N, CA, NH2, NH1, CZ, NE, CD, CG, CB, C, O
    SAVI8: VAL E11: N, CA, CG2, CG1, CB, C, O
    SAVI8: GLN E12: N, CA, NE2, OE1, CD, CG, CB, C, O
    SAVI8: ALA E13: N, CA, CB, C, O
    SAVI8: PRO E14: N, CD, CA, CG, CB, C, O
    SAVI8: ALA E15: N, CA, CB, C, O
    SAVI8: HIS E17: N, CA, CD2, NE2, CE1, ND1, CG, CB, C, O
    SAVI8: ASN E18: N, CA, ND2, OD1, CG, CB, C, O
    SAVI8: THR E22: N, CA, CG2, OG1, CB, C, O
    SAVI8: THR E38: N, CA, CG2, OG1, CB, C, O
    SAVI8: HIS E39: N, CA, CD2, NE2, CE1, ND1, CG, CB, C, O
    SAVI8: PRO E40: N, CD, CA, CG, CB, C, O
    SAVI8: LEU E42: N, CA, CD2, CD1, CG, CB, C, O
    SAVI8: ASN E43: N, CA, ND2, OD1, CG, CB, C, O
    SAVI8: ALA E73: N, CA, CB, C, O
    SAVI8: ALA E74: N, CA, CB, C, O
    SAVI8: LEU E75: N, CA, CD2, CD1, CG, CB, C, O
    SAVI8: ASN E76: N, CA, ND2, OD1, CG, CB, C, O
    SAVI8: LEU E82: N, CA, CD2, CD1, CG, CB, C, O
    SAVI8: GLY E83: N, CA, C, O
    SAVI8: VAL E84: N, CA, CG2, CG1, CB, C, O
    SAVI8: ALA E85: N, CA, CB, C, O
    SAVI8: PRO E86: N, CD, CA, CG, CB, C, O
    SAVI8: SER E103: N, CA, OG, CB, C, O
    SAVI8: VAL E104: N, CA, CG2, CG1, CB, C, O
    SAVI8: SER E105: N, CA, OG, CB, C, O
    SAVI8: ALA E108: N, CA, CB, C, O
    SAVI8: GLN E109: N, CA, NE2, OE1, CD, CG, CB, C, O
    SAVI8: LEU E111: N, CA, CD2, CD1, CG, CB, C, O
    SAVI8: GLU E112: N, CA, OE2, OE1, CD, CG, CB, C, O
    SAVI8: GLY E115: N, CA, C, O
    SAVI8: ASN E116: N, CA, ND2, OD1, CG, CB, C, O
    SAVI8: ALA E122: N, CA, CB, C, O
    SAVI8: SER E128: N, CA, OG, CB, C, O
    SAVI8: PRO E129: N, CD, CA, CG, CB, C, O
    SAVI8: SER E130: N, CA, OG, CB, C, O
    SAVI8: PRO E131: N, CD, CA, CG, CB, C, O
    SAVI8: SER E132: N, CA, OG, CB, C, O
    SAVI8: ALA E133: N, CA, CB, C, O
    SAVI8: THR E134: N, CA, CG2, OG1, CB, C, O
    SAVI8: LEU E135: N, CA, CD2, CD1, CG, CB, C, O
    SAVI8: GLU E136: N, CA, OE2, OE1, CD, CG, CB, C, O
    SAVI8: GLN E137: N, CA, NE2, OE1, CD, CG, CB, C, O
    SAVI8: ALA E138: N, CA, CB, C, O
    SAVI8: VAL E139: N, CA, CG2, CG1, CB, C, O
    SAVI8: ASN E140: N, CA, ND2, OD1, CG, CB, C, O
    SAVI8: SER E141: N, CA, OG, CB, C, O
    SAVI8: ALA E142: N, CA, CB, C, O
    SAVI8: THR E143: N, CA, CG2, OG1, CB, C, O
    SAVI8: SER E144: N, CA, OG, CB, C, O
    SAVI8: VAL E149: N, CA, CG2, CG1, CB, C, O
    SAVI8: VAL E150: N, CA, CG2, CG1, CB, C, O
    SAVI8: SER E156: N, CA, OG, CB, C, O
    SAVI8: GLY E157: N, CA, C, O
    SAVI8: ALA E160: N, CA, CB, C, O
    SAVI8: GLY E161: N, CA, C, O
    SAVI8: SER E162: N, CA, OG, CB, C, O
    SAVI8: ILE E165: N, CA, CD1, CG1, CB, CG2, C, O
    SAVI8: SER E166: N, CA, OG, CB, C, O
    SAVI8: TYR E167: N, CA, OH, CZ, CD2, CE2, CE1, CD1, CG, CB, C, O
    SAVI8: PRO E168: N, CD, CA, CG, CB, C, O
    SAVI8: ARG E170: N, CA, NH2, NH1, CZ, NE, CD, CG, CB, C, O
    SAVI8: TYR E171: N, CA, OH, CZ, CD2, CE2, CE1, CD1, CG, CB, C, O
    SAVI8: ASN E173: N, CA, ND2, OD1, CG, CB, C, O
    SAVI8: THR E180: N, CA, CG2, OG1, CB, C, O
    SAVI8: ASP E181: N, CA, OD2, OD1, CG, CB, C, O
    SAVI8: GLN E182: N, CA, NE2, OE1, CD, CG, CB, C, O
    SAVI8: ASN E183: N, CA, ND2, OD1, CG, CB, C, O
    SAVI8: ASN E184: N, CA, ND2, OD1, CG, CB, C, O
    SAVI8: ASN E185: N, CA, ND2, OD1, CG, CB, C, O
    SAVI8: ARG E186: N, CA, NH2, NH1, CZ, NE, CD, CG, CB, C, O
    SAVI8: ALA E187: N, CA, CB, C, O
    SAVI8: SER E188: N, CA, OG, CB, C, O
    SAVI8: SER E190: N, CA, OG, CB, C, O
    SAVI8: GLN E191: N, CA, NE2, OE1, CD, CG, CB, C, O
    SAVI8: TYR E192: N, CA, OH, CZ, CD2, CE2, CE1, CD1, CG, CB, C, O
    SAVI8: ALA E200: N, CA, CB, C, O
    SAVI8: VAL E203: N, CA, CG2, CG1, CB, C, O
    SAVI8: ASN E204: N, CA, ND2, OD1, CG, CB, C, O
    SAVI8: GLN E206: N, CA, NE2, OE1, CD, CG, CB, C, O
    SAVI8: GLY E211: N, CA, C, O
    SAVI8: SER E212: N, CA, OG, CB, C, O
    SAVI8: THR E213: N, CA, CG2, OG1, CB, C, O
    SAVI8: ALA E215: N, CA, CB, C, O
    SAVI8: SER E216: N, CA, OG, CB, C, O
    SAVI8: VAL E227: N, CA, CG2, CG1, CB, C, O
    SAVI8: ALA E228: N, CA, CB, C, O
    SAVI8: GLY E229: N, CA, C, O
    SAVI8: ALA E230: N, CA, CB, C, O
    SAVI8: THR E255: N, CA, CG2, OG1, CB, C, O
    SAVI8: SER E256: N, CA, OG, CB, C, O
    SAVI8: LEU E257: N, CA, CD2, CD1, CG, CB, C, O
    SAVI8: GLY E258: N, CA, C, O
    SAVI8: SER E259: N, CA, OG, CB, C, O
    SAVI8: ASN E261: N, CA, ND2, OD1, CG, CB, C, O
    SAVI8: LEU E262: N, CA, CD2, CD1, CG, CB, C, O
    SAVI8: LEU E267: N, CA, CD2, CD1, CG, CB, C, O
    SAVI8: VAL E268: N, CA, CG2, CG1, CB, C, O
    SAVI8: ASN E269: N, CA, ND2, OD1, CG, CB, C, O
    Subset SUB5B:
    sub5bmole.list
    Subset SUB5B:
    SAVI8: E2-E4, E16, E19-E21, E23-E24, E28, E37, E41, E44-E45,
    E77-E81, E87-E88,
    SAVI8: E90, E113-E114, E117-E118, E120-E121, E145-E148, E169, E172, E174-E176,
    SAVI8: E193-E196, E198-E199, E214, E231-E234, E236, E243, E247, E250, E253-E254,
    SAVI8: E260, E263-E266, E270-E273, M276H-M277H
    sub5batom.list
    Subset SUB5B:
    SAVI8: GLN E2: N, CA, NE2, OE1, CD, CG, CB, C, O
    SAVI8: SER E3: N, CA, OG, CB, C, O
    SAVI8: VAL E4: N, CA, CG2, CG1, CB, C, O
    SAVI8: ALA E16: N, CA, CB, C, O
    SAVI8: ARG E19: N, CA, NH2, NH1, CZ, NE, CD, CG, CB, C, O
    SAVI8: GLY E20: N, CA, C, O
    SAVI8: LEU E21: N, CA, CD2, CD1, CG, CB, C, O
    SAVI8: GLY E23: N, CA, C, O
    SAVI8: SER E24: N, CA, OG, CB, C, O
    SAVI8: VAL E28: N, CA, CG2, CG1, CB, C, O
    SAVI8: SER E37: N, CA, OG, CB, C, O
    SAVI8: ASP E41: N, CA, OD2, OD1, CG, CB, C, O
    SAVI8: ILE E44: N, CA, CD1, CG1, CB, CG2, C, O
    SAVI8: ARG E45: N, CA, NH2, NH1, CZ, NE, CD, CG, CB, C, O
    SAVI8: ASN E77: N, CA, ND2, OD1, CG, CB, C, O
    SAVI8: SER E78: N, CA, OG, CB, C, O
    SAVI8: ILE E79: N, CA, CD1, CG1, CB, CG2, C, O
    SAVI8: GLY E80: N, CA, C, O
    SAVI8: VAL E81: N, CA, CG2, CG1, CB, C, O
    SAVI8: SER E87: N, CA, OG, CB, C, O
    SAVI8: ALA E88: N, CA, CB, C, O
    SAVI8: LEU E90: N, CA, CD2, CD1, CG, CB, C, O
    SAVI8: TRP E113: N, CA, CD2, CE2, NE1, CD1, CG, CE3, CZ3, CH2, CZ2, CB, C, O
    SAVI8: ALA E114: N, CA, CB, C, O
    SAVI8: ASN E117: N, CA, ND2, OD1, CG, CB, C, O
    SAVI8: GLY E118: N, CA, C, O
    SAVI8: HIS E120: N, CA, CD2, NE2, CE1, ND1, CG, CB, C, O
    SAVI8: VAL E121: N, CA, CG2, CG1, CB, C, O
    SAVI8: ARG E145: N, CA, NH2, NH1, CZ, NE, CD, CG, CB, C, O
    SAVI8: GLY E146: N, CA, C, O
    SAVI8: VAL E147: N, CA, CG2, CG1, CB, C, O
    SAVI8: LEU E148: N, CA, CD2, CD1, CG, CB, C, O
    SAVI8: ALA E169: N, CA, CB, C, O
    SAVI8: ALA E172: N, CA, CB, C, O
    SAVI8: ALA E174: N, CA, CB, C, O
    SAVI8: MET E175: N, CA, CE, SD, CG, CB, C, O
    SAVI8: ALA E176: N, CA, CB, C, O
    SAVI8: GLY E193: N, CA, C, O
    SAVI8: ALA E194: N, CA, CB, C, O
    SAVI8: GLY E195: N, CA, C, O
    SAVI8: LEU E196: N, CA, CD2, CD1, CG, CB, C, O
    SAVI8: ILE E198: N, CA, CD1, CG1, CB, CG2, C, O
    SAVI8: VAL E199: N, CA, CG2, CG1, CB, C, O
    SAVI8: TYR E214: N, CA, OH, CZ, CD2, CE2, CE1, CD1, CG, CB, C, O
    SAVI8: ALA E231: N, CA, CB, C, O
    SAVI8: ALA E232: N, CA, CB, C, O
    SAVI8: LEU E233: N, CA, CD2, CD1, CG, CB, C, O
    SAVI8: VAL E234: N, CA, CG2, CG1, CB, C, O
    SAVI8: GLN E236: N, CA, NE2, OE1, CD, CG, CB, C, O
    SAVI8: ASN E243: N, CA, ND2, OD1, CG, CB, C, O
    SAVI8: ARG E247: N, CA, NH2, NH1, CZ, NE, CD, CG, CB, C, O
    SAVI8: LEU E250: N, CA, CD2, CD1, CG, CB, C, O
    SAVI8: THR E253: N, CA, CG2, OG1, CB, C, O
    SAVI8: ALA E254: N, CA, CB, C, O
    SAVI8: THR E260: N, CA, CG2, OG1, CB, C, O
    SAVI8: TYR E263: N, CA, OH, CZ, CD2, CE2, CE1, CD1, CG, CB, C, O
    SAVI8: GLY E264: N, CA, C, O
    SAVI8: SER E265: N, CA, OG, CB, C, O
    SAVI8: GLY E266: N, CA, C, O
    SAVI8: ALA E270: N, CA, CB, C, O
    SAVI8: GLU E271: N, CA, OE2, OE1, CD, CG, CB, C, O
    SAVI8: ALA E272: N, CA, CB, C, O
    SAVI8: ALA E273: N, CA, CB, C, O
    SAVI8: ION M276H: CA
    SAVI8: ION M277H: CA
    Subset ACTSITE:
    actsitemole.list
    Subset ACTSITE:
    SAVI8: E29-E35, E48-E51, E54, E58-E72, E91-E102, E106-E107, E110, E123-E127,
    SAVI8: E151-E155, E177-E179, E189, E201-E202, E205, E207-E210, E217-E226
    actsiteatom.list
    Subset ACTSITE:
    SAVI8: ALA E29: N, CA, CB, C, O
    SAVI8: VAL E30: N, CA, CG2, CG1, CB, C, O
    SAVI8: LEU E31: N, CA, CD2, CD1, CG, CB, C, O
    SAVI8: ASP E32: N, CA, OD2, OD1, CG, CB, C, O
    SAVI8: THR E33: N, CA, CG2, OG1, CB, C, O
    SAVI8: GLY E34: N, CA, C, O
    SAVI8: ILE E35: N, CA, CD1, CG1, CB, CG2, C, O
    SAVI8: ALA E48: N, CA, CB, C, O
    SAVI8: SER E49: N, CA, OG, CB, C, O
    SAVI8: PHE E50: N, CA, CD2, CE2, CZ, CE1, CD1, CG, CB, C, O
    SAVI8: VAL E51: N, CA, CG2, CG1, CB, C, O
    SAVI8: GLU E54: N, CA, OE2, OE1, CD, CG, CB, C, O
    SAVI8: THR E58: N, CA, CG2, OG1, CB, C, O
    SAVI8: GLN E59: N, CA, NE2, OE1, CD, CG, CB, C, O
    SAVI8: ASP E60: N, CA, OD2, OD1, CG, CB, C, O
    SAVI8: GLY E61: N, CA, C, O
    SAVI8: ASN E62: N, CA, ND2, OD1, CG, CB, C, O
    SAVI8: GLY E63: N, CA, C, O
    SAVI8: HIS E64: N, CA, CD2, NE2, CE1, ND1, CG, CB, C, O
    SAVI8: GLY E65: N, CA, C, O
    SAVI8: THR E66: N, CA, CG2, OG1, CB, C, O
    SAVI8: HIS E67: N, CA, CD2, NE2, CE1, ND1, CG, CB, C, O
    SAVI8: VAL E68: N, CA, CG2, CG1, CB, C, O
    SAVI8: ALA E69: N, CA, CB, C, O
    SAVI8: GLY E70: N, CA, C, O
    SAVI8: THR E71: N, CA, CG2, OG1, CB, C, O
    SAVI8: ILE E72: N, CA, CD1, CG1, CB, CG2, C, O
    SAVI8: TYR E91: N, CA, OH, CZ, CD2, CE2, CE1, CD1, CG, CB, C, O
    SAVI8: ALA E92: N, CA, CB, C, O
    SAVI8: VAL E93: N, CA, CG2, CG1, CB, C, O
    SAVI8: LYS E94: N, CA, NZ, CE, CD, CG, CB, C, O
    SAVI8: VAL E95: N, CA, CG2, CG1, CB, C, O
    SAVI8: LEU E96: N, CA, CD2, CD1, CG, CB, C, O
    SAVI8: GLY E97: N, CA, C, O
    SAVI8: ALA E98: N, CA, CB, C, O
    SAVI8: SER E99: N, CA, OG, CB, C, O
    SAVI8: GLY E100: N, CA, C, O
    SAVI8: SER E101: N, CA, OG, CB, C, O
    SAVI8: GLY E102: N, CA, C, O
    SAVI8: SER E106: N, CA, OG, CB, C, O
    SAVI8: ILE E107: N, CA, CD1, CG1, CB, CG2, C, O
    SAVI8: GLY E110: N, CA, C, O
    SAVI8: ASN E123: N, CA, ND2, OD1, CG, CB, C, O
    SAVI8: LEU E124: N, CA, CD2, CD1, CG, CB, C, O
    SAVI8: SER E125: N, CA, OG, CB, C, O
    SAVI8: LEU E126: N, CA, CD2, CD1, CG, CB, C, O
    SAVI8: GLY E127: N, CA, C, O
    SAVI8: ALA E151: N, CA, CB, C, O
    SAVI8: ALA E152: N, CA, CB, C, O
    SAVI8: SER E153: N, CA, OG, CB, C, O
    SAVI8: GLY E154: N, CA, C, O
    SAVI8: ASN E155: N, CA, ND2, OD1, CG, CB, C, O
    SAVI8: VAL E177: N, CA, CG2, CG1, CB, C, O
    SAVI8: GLY E178: N, CA, C, O
    SAVI8: ALA E179: N, CA, CB, C, O
    SAVI8: PHE E189: N, CA, CD2, CE2, CZ, CE1, CD1, CG, CB, C, O
    SAVI8: PRO E201: N, CD, CA, CG, CB, C, O
    SAVI8: GLY E202: N, CA, C, O
    SAVI8: VAL E205: N, CA, CG2, CG1, CB, C, O
    SAVI8: SER E207: N, CA, OG, CB, C, O
    SAVI8: THR E208: N, CA, CG2, OG1, CB, C, O
    SAVI8: TYR E209: N, CA, OH, CZ, CD2, CE2, CE1, CD1, CG, CB, C, O
    SAVI8: PRO E210: N, CD, CA, CG, CB, C, O
    SAVI8: LEU E217: N, CA, CD2, CD1, CG, CB, C, O
    SAVI8: ASN E218: N, CA, ND2, OD1, CG, CB, C, O
    SAVI8: GLY E219: N, CA, C, O
    SAVI8: THR E220: N, CA, CG2, OG1, CB, C, O
    SAVI8: SER E221: N, CA, OG, CB, C, O
    SAVI8: MET E222: N, CA, CE, SD, CG, CB, C, O
    SAVI8: ALA E223: N, CA, CB, C, O
    SAVI8: THR E224: N, CA, CG2, OG1, CB, C, O
    SAVI8: PRO E225: N, CD, CA, CG, CB, C, O
    SAVI8: HIS E226: N, CA, CD2, NE2, CE1, ND1, CG, CB, C, O
    Subset RESTx:
    restxmole.list
    Subset RESTX:
    NEWMODEL: E5, E13-E14, E22, E38-E40, E42, E73-E76, E82-E86, E103-E105,
    NEWMODEL: E108, E122, E133-E135, E137-E140, E149-E150, E173, E204, E206,
    NEWMODEL: E211-E213, E215-E216, E227-E229, E258, E269
    restxatom.list
    Subset RESTX:
    NEWMODEL: PRO E5: N, CD, CA, CG, CB, C, O
    NEWMODEL: ALA E13: N, CA, CB, C, O
    NEWMODEL: PRO E14: N, CD, CA, CG, CB, C, O
    NEWMODEL: THR E22: N, CA, CG2, OG1, CB, C, O
    NEWMODEL: THR E38: N, CA, CG2, OG1, CB, C, O
    NEWMODEL: HIS E39: N, CA, CD2, NE2, CE1, ND1, CG, CB, C, O
    NEWMODEL: PRO E40: N, CD, CA, CG, CB, C, O
    NEWMODEL: LEU E42: N, CA, CD2, CD1, CG, CB, C, O
    NEWMODEL: ALA E73: N, CA, CB, C, O
    NEWMODEL: ALA E74: N, CA, CB, C, O
    NEWMODEL: LEU E75: N, CA, CD2, CD1, CG, CB, C, O
    NEWMODEL: ASN E76: N, CA, ND2, CD1, CG, CB, C, O
    NEWMODEL: LEU E82: N, CA, CD2, CD1, CG, CB, C, O
    NEWMODEL: GLY E83: N, CA, C, O
    NEWMODEL: VAL E84: N, CA, CG2, CG1, CB, C, O
    NEWMODEL: ALA E85: N, CA, CB, C, O
    NEWMODEL: PRO E86: N, CD, CA, CG, CB, C, O
    NEWMODEL: SER E103: N, CA, OG, CB, C, O
    NEWMODEL: VAL E104: N, CA, CG2, CG1, CB, C, O
    NEWMODEL: SER E105: N, CA, OG, CB, C, O
    NEWMODEL: ALA E108: N, CA, CB, C, O
    NEWMODEL: ALA E122: N, CA, CB, C, O
    NEWMODEL: ALA E133: N, CA, CB, C, O
    NEWMODEL: THR E134: N, CA, CG2, OG1, CB, C, O
    NEWMODEL: LEU E135: N, CA, CD2, CD1, CG, CB, C, O
    NEWMODEL: GLN E137: N, CA, NE2, OE1, CD, CG, CB, C, O
    NEWMODEL: ALA E138: N, CA, CB, C, O
    NEWMODEL: VAL E139: N, CA, CG2, CG1, CB, C, O
    NEWMODEL: ASN E140: N, CA, ND2, OD1, CG, CB, C, O
    NEWMODEL: VAL E149: N, CA, CG2, CG1, CB, C, O
    NEWMODEL: VAL E150: N, CA, CG2, CG1, CB, C, O
    NEWMODEL: ASN E173: N, CA, ND2, OD1, CG, CB, C, O
    NEWMODEL: ASN E204: N, CA, ND2, OD1, CG, CB, C, O
    NEWMODEL: GLN E206: N, CA, NE2, OE1, CD, CG, CB, C, O
    NEWMODEL: GLY E211: N, CA, C, O
    NEWMODEL: SER E212: N, CA, OG, CB, C, O
    NEWMODEL: THR E213: N, CA, CG2, OG1, CB, C, O
    NEWMODEL: ALA E215: N, CA, CB, C, O
    NEWMODEL: SER E216: N, CA, OG, CB, C, O
    NEWMODEL: VAL E227: N, CA, CG2, CG1, CB, C, O
    NEWMODEL: ALA E228: N, CA, CB, C, O
    NEWMODEL: GLY E229: N, CA, C, O
    NEWMODEL: GLY E258: N, CA, C, O
    NEWMODEL: ASN E269: N, CA, ND2, OD1, CG, CB, C, O
    • Example 3
  • Suitable Substitutions in PD498 for Addition of Carboxylic Acid Attachment Groups (—COOH)
  • The 3D structure of PD498 was modeled as described in Example 1. Suitable locations for addition of carboxylic attachment groups (aspartatic acids and glutamic acids) were found as follows. The procedure described in Example 1 was followed. The commands performed in Insight (BIOSYM) are shown in the command files makeDEzone.bcl and makeDEzone2.bcl below:
  • Conservative Substutitions:
    • makeDEzone.bcl
    • Delete Subset *
    • Color Molecule Atoms * Specified Specification 255,0,255
    • Zone Subset ASP :asp:od* Static monomer/residue 10 Color_Subset 255,255,0
    • Zone Subset GLU :glu:oe* Static monomer/residue 10 Color_Subset 255,255,0
    • #NOTE: editnextline C-terminal residue number according to the protein
    • Zone Subset CTERM :280:O Static monomer/residue 10 Color_Subset 255,255,0
    • #NOTE: editnextline ACTSITE residues according to the protein
    • Zone Subset ACTSITE :39,72,226 Static monomer/residue 8 Color_Subset 255,255,0
    • Combine Subset ALLZONE Union ASP GLU
    • Combine Subset ALLZONE Union ALLZONE CTERM
    • Combine Subset ALLZONE Union ALLZONE ACTSITE
    • #NOTE: editnextline object name according to the protein
    • Combine Subset REST Difference PD498FINALMODEL ALLZONE
    • List Subset REST Atom Output_File restatom.list
    • List Subset REST monomer/residue Output_File restmole.list
    • Color Molecule Atoms ACTSITE Specified Specification 255,0,0
    • List Subset ACTSITE Atom Output_File actsiteatom.list
    • List Subset ACTSITE monomer/residue Output_File actsitemole.list
    • #
    • Zone Subset REST5A REST Static Monomer/Residue 5-Color_Subset
    • Combine Subset SUB5A Difference REST5A ACTSITE
    • Combine Subset SUB5B Difference SUB5A REST
    • Color Molecule Atoms SUB5B Specified Specification 255,255,255
    • List Subset SUB5B Atom Output_File sub5batom.list
    • List Subset SUB5B monomer/residue Output_File sub5bmole.list
    • #Now identify sites for asn->asp & gln->glu substitutions and . . .
    • #continue with makezone2.bcl.
    • #Use grep command to identify asn/gln in restatom.list . . .
    • #sub5batom.list & accsiteatom.list.
      Comments:
  • The subset REST contains Gln33 and Asn245, SUB5B contains Gln12, Gln126, Asn209, Gln242, Asn246, Gln248 and Asn266, all of which are solvent exposed.
  • The substitutions Q12E or Q12D, Q33E or Q33D, Q126E or Q126D, N209D or N209E, Q242E or Q242D, N245D or N245E, N246D or N246E, Q248E or Q248D and N266D or N266E are identified in PD498 as sites for mutagenesis within the scope of this invention. Residues are substituted below in section 2, and further analysis done:
  • Non-Conservative Substitutions:
    • makeDEzone2.bcl
    • #sourcefile makezone2.bcl Claus von der Osten 961128
    • #
    • #having scanned lists (grep gln/asn command) and identified sites for
    • #asn->asp & gln->glu substitutions
    • #NOTE: editnextline object name according to protein
    • Copy Object -To_Clipboard -Displace PD498FINALMODEL newmodel
    • Biopolymer
    • #NOTE: editnextline object name according to protein
    • Blank Object On PD498FINALMODEL
    • #NOTE: editnextlines with asn->asp & gln->glu positions
    • Replace Residue newmodel:33 glu L
    • Replace Residue newmodel:245 asp L
    • Replace Residue newmodel:12 glu L
    • Replace Residue newmodel:126 glu L
    • Replace Residue newmodel:209 asp L
    • Replace Residue newmodel:242 glu L
    • Replace Residue newmodel:246 asp L
    • Replace Residue newmodel:248 glu L
    • Replace Residue newmodel:266 asp L
    • #
    • #Now repeat analysis done prior to asn->asp & gln->glu,
    • #now including introduced asp & glu
    • Color Molecule Atoms newmodel Specified Specification 255,0,255
    • Zone Subset ASPx newmodel:asp:od* Static monomer/residue 10 Color_Subset 255,255,0
    • Zone Subset GLUx newmodel:glu:oe* Static monomer/residue 10 Color_Subset 255,255,0
    • #NOTE: editnextline C-terminal residue number according to the protein
    • Zone Subset CTERMx newmodel:280:O Static monomer/residue 10
    • Color_Subset 255,255,0
    • #NOTE: editnextline ACTSITEx residues according to the protein
    • Zone Subset ACTSITEx newmodel:39,72,226 Static monomer/residue 8 Color_Subset 255,255,0
    • Combine Subset ALLZONEx Union ASPx GLUx
    • Combine Subset ALLZONEx Union ALLZONEx CTERMx
    • Combine Subset ALLZONEx Union ALLZONEx ACTSITEx
    • Combine Subset RESTx Difference newmodel ALLZONEx
    • List Subset RESTx Atom Output_File restxatom.list
    • List Subset RESTx monomer/residue Output_File restxmole.list
    • #
    • Color Molecule Atoms ACTSITEx Specified Specification 255,0,0
    • List Subset ACTSITEx Atom Output_File actsitexatom.list
    • List Subset ACTSITEx monomer/residue Output_File actsitexmole.list
    • #
    • #read restxatom.list or restxmole.list to identify sites for (not_gluasp)->gluasp . . .
    • #subst. if needed.
      Comments:
  • The subset RESTx contains only two residues: A233 and G234, none of which are solvent exposed. No further mutagenesis is required to obtain complete protection of the surface. However, it may be necessary to remove some of the reactive carboxylic groups in the active site region to ensure access to the active site of PD498. Acidic residues within the subset ACTSITE are: D39, D58, D68 and D106. Of these only the two latter are solvent exposed and D39 is a functional residue. The mutations D68N, D68Q, D106N and D106Q were found suitable according to the present invention.
  • Relevant Data for Example 3:
  • Solvent Accessibility Data for PD498MODEL: see Example 1 Above.
    Subset REST:
    restmole.list
    Subset REST:
    PD498FINALMODEL: 10-11, 33-35, 54-55, 129-130, 221, 233-234, 236, 240, 243,
    PD498FINALMODEL: 245, 262, 264-265
    restatom.list
    Subset REST:
    PD498FINALMODEL: ALA 10: N, CA, C, O, CB
    PD498FINALMODEL: TYR 11: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ, OH
    PD498FINALMODEL: GLN 33: N, CA, C, O, CB, CG, CD, OE1, NE2
    PD498FINALMODEL: THR 34: N, CA, C, O, CB, OG1, CG2
    PD498FINALMODEL: VAL 35: N, CA, C, O, CB, CG1, CG2
    PD498FINALMODEL: ILE 54: N, CA, C, O, CB, CG1, CG2, CD1
    PD498FINALMODEL: LYS 55: N, CA, C, O, CB, CG, CD, CE, NZ
    PD498FINALMODEL: LYS 129: N, CA, C, O, CB, CG, CD, CE, NZ
    PD498FINALMODEL: VAL 130: N, CA, C, O, CB, CG1, CG2
    PD498FINALMODEL: TYR 221: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ, OH
    PD498FINALMODEL: ALA 233: N, CA, C, O, CB
    PD498FINALMODEL: GLY 234: N, CA, C, O
    PD498FINALMODEL: ALA 236: N, CA, C, O, CB
    PD498FINALMODEL: ALA 240: N, CA, C, O, CB
    PD498FINALMODEL: GLY 243: N, CA, C, O
    PD498FINALMODEL: ASN 245: N, CA, C, O, CB, CG, OD1, ND2
    PD498FINALMODEL: GLY 262: N, CA, C, O
    PD498FINALMODEL: GLY 264: N, CA, C, O
    PD498FINALMODEL: THR 265: N, CA, C, O, CB, OG1, CG2
    Subset SUB5B:
    sub5bmole.list
    Subset SUB5B:
    PD498FINALMODEL: 6-9, 12-13, 31-32, 51-53, 56, 81, 93-94, 97-99, 122,
    126-128,
    PD498FINALMODEL: 131, 155-157, 159, 197-199, 209, 211, 219-220, 232, 235,
    PD498FINALMODEL: 237-239, 241-242, 244, 246-249, 253, 260-261, 263, 266-268
    sub5batom.list
    Subset SUB5B:
    PD498FINALMODEL: PRO 6: N, CA, CD, C, O, CB, CG
    PD498FINALMODEL: TYR 7: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ, OH
    PD498FINALMODEL: TYR 8: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ, OH
    PD498FINALMODEL: SER 9: N, CA, C, O, CB, OG
    PD498FINALMODEL: GLN 12: N, CA, C, O, CB, CG, CD, OE1, NE2
    PD498FINALMODEL: TYR 13: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ, OH
    PD498FINALMODEL: SER 31: N, CA, C, O, CB, OG
    PD498FINALMODEL: THR 32: N, CA, C, O, CB, OG1, CG2
    PD498FINALMODEL: ARG 51: N, CA, C, O, CB, CG, CD, NE, CZ, NH1, NH2
    PD498FINALMODEL: LYS 52: N, CA, C, O, CB, CG, CD, CE, NZ
    PD498FINALMODEL: VAL 53: N, CA, C, O, CB, CG1, CG2
    PD498FINALMODEL: GLY 56: N, CA, C, O
    PD498FINALMODEL: ALA 81: N, CA, C, O, CB
    PD498FINALMODEL: MET 93: N, CA, C, O, CB, CG, SD, CE
    PD498FINALMODEL: ALA 94: N, CA, C, O, CB
    PD498FINALMODEL: THR 97: N, CA, C, O, CB, OG1, CG2
    PD498FINALMODEL: LYS 98: N, CA, C, O, CB, CG, CD, CE, NZ
    PD498FINALMODEL: ILE 99: N, CA, C, O, CB, CG1, CG2, CD1
    PD498FINALMODEL: TYR 122: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ, OH
    PD498FINALMODEL: GLN 126: N, CA, C, O, CB, CG, CD, OE1, NE2
    PD498FINALMODEL: GLY 127: N, CA, C, O
    PD498FINALMODEL: ALA 128: N, CA, C, O, CB
    PD498FINALMODEL: LEU 131: N, CA, C, O, CB, CG, CD1, CD2
    PD498FINALMODEL: GLY 155: N, CA, C, O
    PD498FINALMODEL: ALA 156: N, CA, C, O, CB
    PD498FINALMODEL: VAL 157: N, CA, C, O, CB, CG1, CG2
    PD498FINALMODEL: VAL 159: N, CA, C, O, CB, CG1, CG2
    PD498FINALMODEL: TYR 197: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ, OH
    PD498FINALMODEL: GLY 198: N, CA, C, O
    PD498FINALMODEL: THR 199: N, CA, C, O, CB, OG1, CG2
    PD498FINALMODEL: ASN 209: N, CA, C, O, CB, CG, OD1, ND2
    PD498FINALMODEL: ALA 211: N, CA, C, O, CB
    PD498FINALMODEL: TYR 219: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ, OH
    PD498FINALMODEL: SER 220: N, CA, C, O, CB, OG
    PD498FINALMODEL: VAL 232: N, CA, C, O, CB, CG1, CG2
    PD498FINALMODEL: LEU 235: N, CA, C, O, CB, CG, CD1, CD2
    PD498FINALMODEL: ALA 237: N, CA, C, O, CB
    PD498FINALMODEL: LEU 238: N, CA, C, O, CB, CG, CD1, CD2
    PD498FINALMODEL: LEU 239: N, CA, C, O, CB, CG, CD1, CD2
    PD498FINALMODEL: SER 241: N, CA, C, O, CB, OG
    PD498FINALMODEL: GLN 242: N, CA, C, O, CB, CG, CD, OE1, NE2
    PD498FINALMODEL: LYS 244: N, CA, C, O, CB, CG, CD, CE, NZ
    PD498FINALMODEL: ASN 246: N, CA, C, O, CB, CG, OD1, ND2
    PD498FINALMODEL: VAL 247: N, CA, C, O, CB, CG1, CG2
    PD498FINALMODEL: GLN 248: N, CA, C, O, CB, CG, CD, OE1, NE2
    PD498FINALMODEL: ILE 249: N, CA, C, O, CB, CG1, CG2, CD1
    PD498FINALMODEL: ILE 253: N, CA, C, O, CB, CG1, CG2, CD1
    PD498FINALMODEL: ILE 260: N, CA, C, O, CB, CG1, CG2, CD1
    PD498FINALMODEL: SER 261: N, CA, C, O, CB, OG
    PD498FINALMODEL: THR 263: N, CA, C, O, CB, OG1, CG2
    PD498FINALMODEL: ASN 266: N, CA, C, O, CB, CG, OD1, ND2
    PD498FINALMODEL: PHE 267: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ
    PD498FINALMODEL: LYS 268: N, CA, C, O, CB, CG, CD, CE, NZ
    Subset ACTSITE:
    actsitemole.list
    Subset ACTSITE:
    PD498FINALMODEL: 36-42, 57-60, 66-80, 100-110, 115-116, 119, 132-136,
    160-164,
    PD498FINALMODEL: 182-184, 194, 206-207, 210, 212-215, 222-231
    actsiteatom.list
    Subset ACTSITE:
    PD498FINALMODEL: ALA 36: N, CA, C, O, CB
    PD498FINALMODEL: VAL 37: N, CA, C, O, CB, CG1, CG2
    PD498FINALMODEL: LEU 38: N, CA, C, O, CB, CG, CD1, CD2
    PD498FINALMODEL: ASP 39: N, CA, C, O, CB, CG, OD1, OD2
    PD498FINALMODEL: SER 40: N, CA, C, O, CB, OG
    PD498FINALMODEL: GLY 41: N, CA, C, O
    PD498FINALMODEL: VAL 42: N, CA, C, O, CB, CG1, CG2
    PD498FINALMODEL: TYR 57: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ, OH
    PD498FINALMODEL: ASP 58: N, CA, C, O, CB, CG, OD1, OD2
    PD498FINALMODEL: PHE 59: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ
    PD498FINALMODEL: ILE 60: N, CA, C, O, CB, CG1, CG2, CD1
    PD498FINALMODEL: PRO 66: N, CA, CD, C, O, CB, CG
    PD498FINALMODEL: MET 67: N, CA, C, O, CB, CG, SD, CE
    PD498FINALMODEL: ASP 68: N, CA, C, O, CB, CG, OD1, OD2
    PD498FINALMODEL: LEU 69: N, CA, C, O, CB, CG, CD1, CD2
    PD498FINALMODEL: ASN 70: N, CA, C, O, CB, CG, OD1, ND2
    PD498FINALMODEL: GLY 71: N, CA, C, O
    PD498FINALMODEL: HIS 72: N, CA, C, O, CB, CG, ND1, CD2, CE1, NE2
    PD498FINALMODEL: GLY 73: N, CA, C, O
    PD498FINALMODEL: THR 74: N, CA, C, O, CB, OG1, CG2
    PD498FINALMODEL: HIS 75: N, CA, C, O, CB, CG, ND1, CD2, CE1, NE2
    PD498FINALMODEL: VAL 76: N, CA, C, O, CB, CG1, CG2
    PD498FINALMODEL: ALA 77: N, CA, C, O, CB
    PD498FINALMODEL: GLY 78: N, CA, C, O
    PD498FINALMODEL: THR 79: N, CA, C, O, CB, OG1, CG2
    PD498FINALMODEL: VAL 80: N, CA, C, O, CB, CG1, CG2
    PD498FINALMODEL: LEU 100: N, CA, C, O, CB, CG, CD1, CD2
    PD498FINALMODEL: ALA 101: N, CA, C, O, CB
    PD498FINALMODEL: VAL 102: N, CA, C, O, CB, CG1, CG2
    PD498FINALMODEL: ARG 103: N, CA, C, O, CB, CG, CD, NE, CZ, NH1, NH2
    PD498FINALMODEL: VAL 104: N, CA, C, O, CB, CG1, CG2
    PD498FINALMODEL: LEU 105: N, CA, C, O, CB, CG, CD1, CD2
    PD498FINALMODEL: ASP 106: N, CA, C, O, CB, CG, OD1, OD2
    PD498FINALMODEL: ALA 107: N, CA, C, O, CB
    PD498FINALMODEL: ASN 108: N, CA, C, O, CB, CG, OD1, ND2
    PD498FINALMODEL: GLY 109: N, CA, C, O
    PD498FINALMODEL: SER 110: N, CA, C, O, CB, OG
    PD498FINALMODEL: SER 115: N, CA, C, O, CB, OG
    PD498FINALMODEL: ILE 116: N, CA, C, O, CB, CG1, CG2, CD1
    PD498FINALMODEL: GLY 119: N, CA, C, O
    PD498FINALMODEL: ASN 132: N, CA, C, O, CB, CG, OD1, ND2
    PD498FINALMODEL: LEU 133: N, CA, C, O, CB, CG, CD1, CD2
    PD498FINALMODEL: SER 134: N, CA, C, O, CB, OG
    PD498FINALMODEL: LEU 135: N, CA, C, O, CB, CG, CD1, CD2
    PD498FINALMODEL: GLY 136: N, CA, C, O
    PD498FINALMODEL: ALA 160: N, CA, C, O, CB
    PD498FINALMODEL: ALA 161: N, CA, C, O, CB
    PD498FINALMODEL: ALA 162: N, CA, C, O, CB
    PD498FINALMODEL: GLY 163: N, CA, C, O
    PD498FINALMODEL: ASN 164: N, CA, C, O, CB, CG, OD1, ND2
    PD498FINALMODEL: VAL 182: N, CA, C, O, CB, CG1, CG2
    PD498FINALMODEL: GLY 183: N, CA, C, O
    PD498FINALMODEL: ALA 184: N, CA, C, O, CB
    PD498FINALMODEL: PHE 194: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ
    PD498FINALMODEL: PRO 206: N, CA, CD, C, O, CB, CG
    PD498FINALMODEL: GLY 207: N, CA, C, O
    PD498FINALMODEL: ILE 210: N, CA, C, O, CB, CG1, CG2, CD1
    PD498FINALMODEL: SER 212: N, CA, C, O, CB, OG
    PD498FINALMODEL: THR 213: N, CA, C, O, CB, OG1, CG2
    PD498FINALMODEL: VAL 214: N, CA, C, O, CB, CG1, CG2
    PD498FINALMODEL: PRO 215: N, CA, CD, C, O, CB, CG
    PD498FINALMODEL: MET 222: N, CA, C, O, CB, CG, SD, CE
    PD498FINALMODEL: SER 223: N, CA, C, O, CB, OG
    PD498FINALMODEL: GLY 224: N, CA, C, O
    PD498FINALMODEL: THR 225: N, CA, C, O, CB, OG1, CG2
    PD498FINALMODEL: SER 226: N, CA, C, O, CB, OG
    PD498FINALMODEL: MET 227: N, CA, C, O, CB, CG, SD, CE
    PD498FINALMODEL: ALA 228: N, CA, C, O, CB
    PD498FINALMODEL: SER 229: N, CA, C, O, CB, OG
    PD498FINALMODEL: PRO 230: N, CA, CD, C, O, CB, CG
    PD498FINALMODEL: HIS 231: N, CA, C, O, CB, CG, ND1, CD2, CE1, NE2
    Subset RESTx:
    restxmole.list
    Subset RESTX:
    NEWMODEL: 233-234
    restxatom.list
    Subset RESTX:
    NEWMODEL: ALA 233: N, CA, C, O, CB
    NEWMODEL: GLY 234: N, CA, C, O
    • Example 4
  • Suitable substitutions in the Arthromyces ramosus peroxidase for addition of carboxylic acid attachment groups (—COOH) Suitable locations for addition of carboxylic attachment groups (aspartatic acids and glutamic acids) in a non-hydrolytic enzyme, Arthromyces ramosus peroxidase were found as follows.
  • The 3D structure of this oxido-reductase is available in the Brookhaven Databank as larp.pdb. This A. ramosus peroxidase contains 344 amino acid residues. The first eight residues are not visible in the X-ray structure: QGPGGGGG, and N143 is glycosylated.
  • The procedure described in Example 1 was followed.
  • The amino acid sequence of Arthromyces ramosus Peroxidase (E.C.1.11.1.7) is shown in SEQ ID NO: 4.
  • The commands performed in Insight (BIOSYM) are shown in the command files makeDEzone.bcl and makeDEzone2.bcl below. The C-terminal residue is P344, the ACTSITE is defined as the heme group and the two histidines coordinating it (H56 & H184).
  • Conservative Substitutions:
    • makeDEzone.bcl
    • Delete Subset *
    • Color Molecule Atoms * Specified Specification 255,0,255
    • Zone Subset ASP :asp:od* Static monomer/residue 10 Color_Subset 255,255,0
    • Zone Subset GLU :glu:oe* Static monomer/residue 10 Color_Subset 255,255,0
    • #NOTE: editnextline C-terminal residue number according to the protein
    • Zone Subset CTERM :344:O Static monomer/residue 10 Color_Subset 255,255,0
    • #NOTE: editnextline ACTSITE residues according to the protein
    • Zone Subset ACTSITE :HEM,56,184 Static monomer/residue 8 Color_Subset 255,255,0
    • Combine Subset ALLZONE Union ASP GLU
    • Combine Subset ALLZONE Union ALLZONE CTERM
    • Combine Subset ALLZONE Union ALLZONE ACTSITE
    • #NOTE: editnextline object name according to the protein
    • Combine Subset REST Difference ARP ALLZONE
    • List Subset REST Atom Output_File restatom.list
    • List Subset REST monomer/residue Output_File restmole.list
    • Color Molecule Atoms ACTSITE Specified Specification 255,0,0
    • List Subset ACTSITE Atom Output_File actsiteatom.list
    • List Subset ACTSITE monomer/residue Output_File actsitemole.list
    • #
    • Zone Subset REST5A REST Static Monomer/Residue 5-Color_Subset
    • Combine Subset SUB5A Difference REST5A ACTSITE
    • Combine Subset SUB5B Difference SUB5A REST
    • Color Molecule Atoms SUB5B Specified Specification 255,255,255
    • List Subset SUB5B Atom Output_File sub5batom.list
    • List Subset SUB5B monomer/residue Output_File sub5bmole.list
    • #Now identify sites for asn->asp & gln->glu substitutions and . . .
    • #continue with makezone2.bcl.
    • #Use grep command to identify asn/gln in restatom.list . . .
    • #sub5batom.list & accsiteatom.list.
      Comments:
  • The subset REST contains Gln70, and SUB5B contains Gln34, Asn128, Asn303 all of which are solvent exposed. The substitutions Q34E or Q34D, Q70E or Q70D, N128D or N128E and N303D or N303E are identified in A. ramosus peroxidase as sites for mutagenesis. Residues are substituted below and further analysis done:
  • Non-Conservative Substitutions:
    • makeDEzone2.bcl
    • #sourcefile makezone2.bcl Claus von der Osten 961128
    • #
    • #having scanned lists (grep gln/asn command) and identified sites for . . .
    • #asn->asp & gln->glu substitutions
    • #NOTE: editnextline object name according to protein
    • Copy Object -To_Clipboard -Displace ARP newmodel
    • Biopolymer
    • #NOTE: editnextline object name according to protein
    • Blank Object On ARP
    • #NOTE: editnextlines with asn->asp & gln->glu positions
    • Replace Residue newmodel:34 glu L
    • Replace Residue newmodel:70 glu L
    • Replace Residue newmodel:128 asp L
    • Replace Residue newmodel:303 asp L
    • #
    • #Now repeat analysis done prior to asn->asp & gln->glu, . . .
    • #now including introduced asp & glu
    • Color Molecule Atoms newmodel Specified Specification 255,0,255
    • Zone Subset ASPx newmodel:asp:od* Static monomer/residue 10 Color_Subset 255,255,0
    • xZone Subset GLUx newmodel:glu:oe* Static monomer/residue 10 Color_Subset 255,255,0
    • #NOTE: editnextline C-terminal residue number according to the protein
    • Zone Subset CTERMx newmodel:344:O Static monomer/residue 10 Color_Subset 255,255,0
    • #NOTE: editnextline ACTSITEx residues according to the protein
    • Zone Subset ACTSITEx newmodel:HEM,56,184 Static monomer/residue 8 Color_Subset 255,255,0
    • Combine Subset ALLZONEx Union ASPx GLUx
    • Combine Subset ALLZONEx Union ALLZONEx CTERMx
    • Combine Subset ALLZONEx Union ALLZONEx ACTSITEx
    • Combine Subset RESTx Difference newmodel ALLZONEx
    • List Subset RESTx Atom Output_File restxatom.list
    • List Subset RESTx monomer/residue Output_File restxmole.list
    • #
    • Color Molecule Atoms ACTSITEx Specified Specification 255,0,0
    • List Subset ACTSITEx Atom Output_File actsitexatom.list
    • List Subset ACTSITEx monomer/residue Output_File actsitexmole.list
    • #
    • #read restxatom.list or restxmole.list to identify sites for (not_gluasp)->gluasp . . .
    • #subst. if needed.
      Comments:
  • The subset RESTx contains only four residues: S9, S334, G335 and P336, all of which are >5% solvent exposed. The mutations S9D, S9E, S334D, S334E, G335D, G335E, P336D and P336E are proposed in A. ramosus peroxidase. Acidic residues within the subset ACTSITE are: E44, D57, D77, E87, E176, D179, E190, D202, D209, D246 and the N-terminal carboxylic acid on P344. Of these only E44, D77, E176, D179, E190, D209, D246 and the N-terminal carboxylic acid on P344 are solvent exposed. Suitable sites for mutations are E44Q, D77N, E176Q, D179N, E190Q, D209N and D246N. D246N and D246E are risky mutations due to D246's importance for binding of heme.
  • The N-terminal 8 residues were not included in the calculations above, as they do not appear in the structure. None of these 8 residues, QGPGGGG, contain carboxylic groups. The following variants are proposed as possible mutations to enable attachment to this region: Q1E, Q1D, G2E, G2D, P3E, P3D, G4E, G4D, G5E, G5D, G6E, G6D, G7E, G7D, G8E, G8D.
  • Relevant Data for Example 4:
  • Solvent accessibility data for A. ramosus peroxidase (Note: as the first eight residues are missing in the X-ray structure, the residue numbers printed in the accessibility list below are 8 lower than those used elsewhere for residue numbering.
    # ARP Thu Jan 30 15:39:05 MET 1997
    # residue area
    SER_1 143.698257
    VAL_2 54.879990
    THR_3 86.932701
    CYS_4 8.303715
    PRO_5 126.854782
    GLY_6 53 .771488
    GLY_7 48.137802
    GLN_8 62.288475
    SER_9 79.932549
    THR_10 16.299215
    SER_11 81.928642
    ASN_12 51.432678
    SER_13 81.993019
    GLN_14 92.344009
    CYS_15 0.000000
    CYS_16 32.317432
    VAL_17 54.067810
    TRP_18 6.451035
    PHE_19 25.852070
    ASP_20 79.033997
    VAL_21 0.268693
    LEU_22 22.032858
    ASP_23 90.111404
    ASP_24 43.993240
    LEU_25 1.074774
    GLN_26 25.589321
    THR_27 82.698059
    ASN_28 96.600883
    PHE_29 32.375275
    TYR_30 5.898365
    GLN_31 103.380585
    GLY_32 40.042034
    SER_33 46.789322
    LYS_34 87.161873
    CYS_35 12.827215
    GLU_36 51.582657
    SER_37 16.378180
    PRO_38 33.560043
    VAL_39 6.448641
    ARG_40 7.068311
    LYS_41 15.291286
    ILE_42 1.612160
    LEU_43 1.880854
    ARG_44 16.906845
    ILE_45 0.000000
    VAL_46 2.312647
    PHE_47 2.955627
    HIS_48 20.392527
    ASP_49 4.238116
    ALA_50 0.510757
    ILE_51 1.576962
    GLY_52 2.858601
    PHE_53 48.633503
    SER_54 8.973248
    PRO_55 58.822315
    ALA_56 59.782852
    LEU_57 46.483955
    THR_58 86.744827
    ALA_59 89.515816
    ALA_60 81.163239
    GLY_61 70.119019
    GLN_62 112.635498
    PHE_63 93.522354
    GLY_64 2.742587
    GLY_65 13.379636
    GLY_66 22.722847
    GLY_67 0.000000
    ALA_68 0.268693
    ASP_69 12.074840
    GLY_70 0.700486
    SER_71 0.000000
    ILE_72 0.000000
    ILE_73 0.000000
    ALA_74 17.304443
    HIS_75 41.071186
    SER_76 20.000793
    ASN_77 120.855316
    ILE_78 66.574982
    GLU_79 2.334954
    LEU_80 41.329689
    ALA_81 77.370575
    PHE_82 38.758774
    PRO_83 131.946289
    ALA_84 34.893864
    ASN_85 5.457000
    GLY_86 43.364151
    GLY_87 51.561348
    LEU_88 0.242063
    THR_89 73.343575
    ASP_90 130.139389
    THR_91 17.863211
    ILE_92 0.268693
    GLU_93 92.210396
    ALA_94 35.445068
    LEU_95 1.343467
    ARG_96 31.175611
    ALA_97 44.650192
    VAL_98 17.698566
    GLY_99 1.471369
    ILE_100 62.441463
    ASN_101 107.139748
    HIS_102 46.952496
    GLY_103 46.559296
    VAL_104 11.342628
    SER_105 15.225677
    PHE_106 6.422011
    GLY_107 3.426864
    ASP_108 10.740790
    LEU_109 0.268693
    ILE_110 1.880854
    GLN_111 31.867456
    PHE_112 0.000000
    ALA_113 0.000000
    THR_114 3.656114
    ALA_115 8.299393
    VAL_116 0.268693
    GLY_117 0.268693
    MET_118 3.761708
    SER_119 14.536770
    ASN_120 25.928799
    CYS_121 0.537387
    PRO_122 29.798336
    GLY_123 33.080013
    SER_124 17.115562
    PRO_125 36.908714
    ARG_126 108.274727
    LEU_127 21.238588
    GLU_128 53.742313
    PHE_129 3.761708
    LEU_130 12.928699
    THR_131 10.414591
    GLY_132 47.266495
    ARG_133 12.247048
    SER_134 63.047237
    ASN_135 31.403708
    SER_136 97.999619
    SER_137 28.505201
    GLN_138 102.845520
    PRO_139 49.691917
    SER_140 9.423104
    PRO_141 25.724171
    PRO_142 80.706665
    SER_143 105.318176
    LEU_144 20.154398
    ILE_145 41.288322
    PRO_146 10.462679
    GLY_147 19.803421
    PRO_148 18.130360
    GLY_149 47.391853
    ASN_150 60.248917
    THR_151 87.887985
    VAL_152 13.870322
    THR_153 74.664734
    ALA_154 45.251106
    ILE_155 2.686934
    LEU_156 28.720940
    ASP_157 110.081253
    ARG_158 31.228874
    MET_159 1.612160
    GLY_160 38.223858
    ASP_161 46.293152
    ALA_162 9.877204
    GLY_163 34.267326
    PHE_164 11.057570
    SER_165 51.158882
    PRO_166 62.767738
    ASP_167 75.164917
    GLU_168 43.334976
    VAL_169 6.365355
    VAL_170 2.955627
    ASP_171 7.004863
    LEU_172 1.880854
    LEU_173 3.197691
    ALA_174 0.000000
    ALA_175 1.074774
    HIS_176 0.502189
    SER_177 0.806080
    LEU_178 3.197691
    ALA_179 3.337480
    SER_180 0.466991
    GLN_181 2.122917
    GLU_182 40.996552
    GLY_183 62.098671
    LEU_184 23.954853
    ASN_185 15.918136
    SER_186 95.185318
    ALA_187 59.075272
    ILE_188 27.675419
    PHE_189 102.799423
    ARG_190 55.265549
    SER_191 6.986028
    PRO_192 2.686934
    LEU_193 12.321225
    ASP_194 2.127163
    SER_195 33.556419
    THR_196 33.049286
    PRO_197 20.874798
    GLN_198 65.729698
    VAL_199 31.705818
    PHE_200 4.753195
    ASP_201 13.744506
    THR_202 1.612160
    GLN_203 16.081930
    PHE_204 2.581340
    TYR_205 1.880854
    ILE_206 9.356181
    GLU_207 0.735684
    THR_208 10.685907
    LEU_209 9.672962
    LEU_210 2.955627
    LYS_211 77.176834
    GLY_212 40.968609
    THR_213 78.718216
    THR_214 21.738384
    GLN_215 77.622299
    PRO_216 25.441587
    GLY_217 8.320850
    PRO_218 96.972305
    SER_219 64.627823
    LEU_220 85.732414
    GLY_221 27.361111
    PHE_222 134.620178
    ALA_223 3.873014
    GLU_224 12.141763
    GLU_225 65.129868
    LEU_226 76.105843
    SER_227 0.268693
    PRO_228 7.017754
    PHE_229 0.000000
    PRO_230 47.827423
    GLY_231 23.790522
    GLU_232 6.643466
    PHE_233 6.713862
    ARG_234 18.012030
    MET_235 4.598188
    ARG_236 91.415581
    SER_237 1.982125
    ASP_238 6.246871
    ALA_239 12.897283
    LEU_240 76.820526
    LEU_241 3.224321
    ALA_242 1.400973
    ARG_243 77.207176
    ASP_244 36.207306
    SER_245 104.023796
    ARG_246 121.852341
    THR_247 2.955627
    ALA_248 4.810700
    CYS_249 47.331306
    ARG_250 62.062778
    TRP_251 2.418241
    GLN_252 5.554953
    SER_253 38.284832
    MET_254 1.124224
    THR_255 0.000000
    SER_256 53.758987
    SER_257 37.276134
    ASN_258 44.381340
    GLU_259 149.565140
    VAL_260 57.500389
    MET_261 2.679314
    GLY_262 10.175152
    GLN_263 107.458916
    ARG_264 36.402130
    TYR_265 0.233495
    ARG_266 91.179619
    ALA_267 53.708500
    ALA_268 6.504294
    MET_269 17.122011
    ALA_270 22.455158
    LYS_271 73.386177
    MET_272 3.959508
    SER_273 15.043281
    VAL_274 23.887930
    LEU_275 17.196379
    GLY_276 44.362202
    PHE_277 68.062485
    ASP_278 94.902039
    ARG_279 113.549011
    ASN_280 134.886017
    ALA_281 72.340973
    LEU_282 26.692348
    THR_283 27.696728
    ASP_284 72.214157
    CYS_285 0.000000
    SER_286 28.209335
    ASP_287 64.560753
    VAL_288 7.040061
    ILE_289 8.665112
    PRO_290 48.682365
    SER_291 86.141670
    ALA_292 29.031240
    VAL_293 84.432014
    SER_294 85.944153
    ASN_295 49.017288
    ASN_296 133.459198
    ALA_297 57.283794
    ALA_298 65.233749
    PRO_299 24.751518
    VAL_300 45.409184
    ILE_301 8.060802
    PRO_302 14.742939
    GLY_303 16.589832
    GLY_304 34.238071
    LEU_305 24.719791
    THR_306 49.356300
    VAL_307 71.491821
    ASP_308 130.906174
    ASP_309 31.733070
    ILE_310 19.581894
    GLU_311 81.414574
    VAL_312 94.769890
    SER_313 39.688896
    CYS_314 9.998511
    PRO_315 120.328018
    SER_316 95.364319
    GLU_317 65.560959
    PRO_318 100.254364
    PHE_319 46.284115
    PRO_320 31.328060
    GLU_321 177.602249
    ILE_322 33.449741
    ALA_323 46.892982
    THR_324 79.976471
    ALA_325 36.423820
    SER_326 124.467422
    GLY_327 28.219524
    PRO_328 107.553696
    LEU_329 86.789825
    PRO_330 34.287163
    SER_331 75.764053
    LEU_332 32.840569
    ALA_333 61.516434
    PRO_334 82.389992
    ALA_335 6.246871
    PRO_336 56.750813
    HEM_337 60.435017
    CA_338 2.078997
    CA_339 0.000000
    NAG_340 141.534668
    NAG_341 186.311371
    Subset REST:
    restmole.list
    Subset REST:
    ARP: 9, 69-70, 125, 127, 133, 299-301, 334-336
    restatom.list
    Subset REST:
    ARP: SER 9: N, CA, C, O, CB, OG
    ARP: GLY 69: N, CA, C, O
    ARP: GLN 70: N, CA, C, O, CB, CG, CD, OE1, NE2
    ARP: GLY 125: N, CA, C, O
    ARP: SER 127: N, CA, C, O, CB, OG
    ARP: PRO 133: N, CA, CD, C, O, CB, CG
    ARP: SER 299: N, CA, C, O, CB, OG
    ARP: ALA 300: N, CA, C, O, CB
    ARP: VAL 301: N, CA, C, O, CB, CG1, CG2
    ARP: SER 334: N, CA, C, O, CB, OG
    ARP: GLY 335: N, CA, C, O
    ARP: PRO 336: N, CA, CD, C, O, CB, CG
    Subset SUB5B:
    sub5bmole.list
    Subset SUB5B:
    ARP: 10-11, 34, 38, 65-68, 71-72, 120-121, 123-124, 128-132, 134, 270, 274,
    ARP: 297-298, 302-303, 311-312, 332-333, 337-338
    sub5batom.list
    Subset SUB5B:
    ARP: VAL 10: N, CA, C, O, CB, CG1, CG2
    ARP: THR 11: N, CA, C, O, CB, OG1, CG2
    ARP: GLN 34: N, CA, C, O, CB, CG, CD, OE1, NE2
    ARP: TYR 38: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ, OH
    ARP: LEU 65: N, CA, C, O, CB, CG, CD1, CD2
    ARP: THR 66: N, CA, C, O, CB, OG1, CG2
    ARP: ALA 67: N, CA, C, O, CB
    ARP: ALA 68: N, CA, C, O, CB
    ARP: PHE 71: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ
    ARP: GLY 72: N, CA, C, O
    ARP: PHE 120: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ
    ARP: ALA 121: N, CA, C, O, CB
    ARP: ALA 123: N, CA, C, O, CB
    ARP: VAL 124: N, CA, C, O, CB, CG1, CG2
    ARP: ASN 128: N, CA, C, O, CB, CG, OD1, ND2
    ARP: CYS 129: N, CA, C, O, CB, SG
    ARP: PRO 130: N, CA, CD, C, O, CB, CG
    ARP: GLY 131: N, CA, C, O
    ARP: SER 132: N, CA, C, O, CB, OG
    ARP: ARG 134: N, CA, C, O, CB, CG, CD, NE, CZ, NH1, NH2
    ARP: GLY 270: N, CA, C, O
    ARP: ARG 274: N, CA, C, O, CB, CG, CD, NE, CZ, NH1, NH2
    ARP: ILE 297: N, CA, C, O, CB, CG1, CG2, CD1
    ARP: PRO 298: N, CA, CD, C, O, CB, CG
    ARP: SER 302: N, CA, C, O, CB, OG
    ARP: ASN 303: N, CA, C, O, CB, CG, OD1, ND2
    ARP: GLY 311: N, CA, C, O
    ARP: GLY 312: N, CA, C, O
    ARP: THR 332: N, CA, C, O, CB, OG1, CG2
    ARP: ALA 333: N, CA, C, O, CB
    ARP: LEU 337: N, CA, C, O, CB, CG, CD1, CD2
    ARP: PRO 338: N, CA, CD, C, O, CB, CG
    Subset ACTSITE:
    actsitemole.list
    Subset ACTSITE:
    ARP: 44-61, 75-77, 79-80, 87-88, 90-96, 99, 118, 122, 126, 135, 148-149, 152-158,
    ARP: 163-164, 167, 176-194, 197-205, 207-209, 211-213, 216, 230-231, 241,
    ARP: 243-246, 249, 259, 273, 277, 280, 343-347H
    actsiteatom.list
    Subset ACTSITE:
    ARP: GLU 44: N, CA, C, O, CB, CG, CD, OE1, OE2
    ARP: SER 45: N, CA, C, O, CB, OG
    ARP: PRO 46: N, CA, CD, C, O, CB, CG
    ARP: VAL 47: N, CA, C, O, CB, CG1, CG2
    ARP: ARG 48: N, CA, C, O, CB, CG, CD, NE, CZ, NH1, NH2
    ARP: LYS 49: N, CA, C, O, CB, CG, CD, CE, NZ
    ARP: ILE 50: N, CA, C, O, CB, CG1, CG2, CD1
    ARP: LEU 51: N, CA, C, O, CB, CG, CD1, CD2
    ARP: ARG 52: N, CA, C, O, CB, CG, CD, NE, CZ, NH1, NH2
    ARP: ILE 53: N, CA, C, O, CB, CG1, CG2, CD1
    ARP: VAL 54: N, CA, C, O, CB, CG1, CG2
    ARP: PHE 55: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ
    ARP: HIS 56: N, CA, C, O, CB, CG, ND1, CD2, CE1, NE2
    ARP: ASP 57: N, CA, C, O, CB, CG, OD1, OD2
    ARP: ALA 58: N, CA, C, O, CB
    ARP: ILE 59: N, CA, C, O, CB, CG1, CG2, CD1
    ARP: GLY 60: N, CA, C, O
    ARP: PHE 61: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ
    ARP: GLY 75: N, CA, C, O
    ARP: ALA 76: N, CA, C, O, CB
    ARP: ASP 77: N, CA, C, O, CB, CG, OD1, OD2
    ARP: SER 79: N, CA, C, O, CB, OG
    ARP: ILE 80: N, CA, C, O, CB, CG1, CG2, CD1
    ARP: GLU 87: N, CA, C, O, CB, CG, CD, OE1, OE2
    ARP: LEU 88: N, CA, C, O, CB, CG, CD1, CD2
    ARP: PHE 90: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ
    ARP: PRO 91: N, CA, CD, C, O, CB, CG
    ARP: ALA 92: N, CA, C, O, CB
    ARP: ASN 93: N, CA, C, O, CB, CG, OD1, ND2
    ARP: GLY 94: N, CA, C, O
    ARP: GLY 95: N, CA, C, O
    ARP: LEU 96: N, CA, C, O, CB, CG, CD1, CD2
    ARP: THR 99: N, CA, C, O, CB, OG1, CG2
    ARP: ILE 118: N, CA, C, O, CB, CG1, CG2, CD1
    ARP: THR 122: N, CA, C, O, CB, OG1, CG2
    ARP: MET 126: N, CA, C, O, CB, CG, SD, CE
    ARP: LEU 135: N, CA, C, O, CB, CG, CD1, CD2
    ARP: SER 148: N, CA, C, O, CB, OG
    ARP: PRO 149: N, CA, CD, C, O, CB, CG
    ARP: LEU 152: N, CA, C, O, CB, CG, CD1, CD2
    ARP: ILE 153: N, CA, C, O, CB, CG1, CG2, CD1
    ARP: PRO 154: N, CA, CD, C, O, CB, CG
    ARP: GLY 155: N, CA, C, O
    ARP: PRO 156: N, CA, CD, C, O, CB, CG
    ARP: GLY 157: N, CA, C, O
    ARP: ASN 158: N, CA, C, O, CB, CG, OD1, ND2
    ARP: ILE 163: N, CA, C, O, CB, CG1, CG2, CD1
    ARP: LEU 164: N, CA, C, O, CB, CG, CD1, CD2
    ARP: MET 167: N, CA, C, O, CB, CG, SD, CE
    ARP: GLU 176: N, CA, C, O, CB, CG, CD, OE1, OE2
    ARP: VAL 177: N, CA, C, O, CB, CG1, CG2
    ARP: VAL 178: N, CA, C, O, CB, CG1, CG2
    ARP: ASP 179: N, CA, C, O, CB, CG, OD1, OD2
    ARP: LEU 180: N, CA, C, O, CB, CG, CD1, CD2
    ARP: LEU 181: N, CA, C, O, CB, CG, CD1, CD2
    ARP: ALA 182: N, CA, C, O, CB
    ARP: ALA 183: N, CA, C, O, CB
    ARP: HIS 184: N, CA, C, O, CB, CG, ND1, CD2, CE1, NE2
    ARP: SER 185: N, CA, C, O, CB, OG
    ARP: LEU 186: N, CA, C, O, CB, CG, CD1, CD2
    ARP: ALA 187: N, CA, C, O, CB
    ARP: SER 188: N, CA, C, O, CB, OG
    ARP: GLN 189: N, CA, C, O, CB, CG, CD, OE1, NE2
    ARP: GLU 190: N, CA, C, O, CB, CG, CD, OE1, OE2
    ARP: GLY 191: N, CA, C, O
    ARP: LEU 192: N, CA, C, O, CB, CG, CD1, CD2
    ARP: ASN 193: N, CA, C, O, CB, CG, OD1, ND2
    ARP: SER 194: N, CA, C, O, CB, OG
    ARP: PHE 197: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ
    ARP: ARG 198: N, CA, C, O, CB, CG, CD, NE, CZ, NH1, NH2
    ARP: SER 199: N, CA, C, O, CB, OG
    ARP: PRO 200: N, CA, CD, C, O, CB, CG
    ARP: LEU 201: N, CA, C, O, CB, CG, CD1, CD2
    ARP: ASP 202: N, CA, C, O, CB, CG, OD1, OD2
    ARP: SER 203: N, CA, C, O, CB, OG
    ARP: THR 204: N, CA, C, O, CB, OG1, CG2
    ARP: PRO 205: N, CA, CD, C, O, CB, CG
    ARP: VAL 207: N, CA, C, O, CB, CG1, CG2
    ARP: PHE 208: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ
    ARP: ASP 209: N, CA, C, O, CB, CG, OD1, OD2
    ARP: GLN 211: N, CA, C, O, CB, CG, CD, OE1, NE2
    ARP: PHE 212: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ
    ARP: TYR 213: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ, OH
    ARP: THR 216: N, CA, C, O, CB, OG1, CG2
    ARP: PHE 230: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ
    ARP: ALA 231: N, CA, C, O, CB
    ARP: PHE 241: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ
    ARP: MET 243: N, CA, C, O, CB, CG, SD, CE
    ARP: ARG 244: N, CA, C, O, CB, CG, CD, NE, CZ, NH1, NH2
    ARP: SER 245: N, CA, C, O, CB, OG
    ARP: ASP 246: N, CA, C, O, CB, CG, OD1, OD2
    ARP: LEU 249: N, CA, C, O, CB, CG, CD1, CD2
    ARP: TRP 259: N, CA, C, O, CB, CG, CD1, CD2, NE1, CE2, CE3, CZ2, CZ3, CH2
    ARP: TYR 273: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ, OH
    ARP: MET 277: N, CA, C, O, CB, CG, SD, CE
    ARP: MET 280: N, CA, C, O, CB, CG, SD, CE
    ARP: ALA 343: N, CA, C, O, CB
    ARP: PRO 344: N, CA, CD, C, O, OXT, CB, CG
    ARP: HEM 345H: FE, NA, NB, NC, ND, CHA, CHB, CHC, CHD, C1A, C2A, C3A, C4A, CMA,
    CAA, CBA, CGA
    ARP: HEM 345H: O1A, O2A, C1B, C2B, C3B, C4B, CMB, CAB, CBB, C1C, C2C, C3C, C4C,
    CMC, CAC, CBC
    ARP: HEM 345H: C1D, C2D, C3D, C4D, CMD, CAD, CBD, CGD, O1D, O2D
    ARP: CA 346H: CA
    ARP: CA 347H: CA
    Subset RESTx:
    restxmole.list
    Subset RESTX
    NEWMODEL: 9, 334-336
    restxatom.list
    Subset RESTX:
    NEWMODEL: SER 9: N, CA, C, O, CB, OG
    NEWMODEL: SER 334: N, CA, C, O, CB, OG
    NEWMODEL: GLY 335: N, CA, C, O
    NEWMODEL: PRO 336: N, CA, CD, C, O, CB, CG
    • Example 5
  • Activation of mPEG 15,000 with N-succinimidyl Carbonate
  • mPEG 15,000 was suspended in toluene (4 ml/g of mPEG) 20% was distilled off at normal pressure to dry the reactants azeotropically. Dichloromethane (dry 1 ml/g mPEG) was added when the solution was cooled to 30° C. and phosgene in toluene (1.93 M 5 mole/mole mPEG) was added and mixture stirred at room temperature overnight. The mixture was evaporated to dryness and the desired product was obtained as waxy lumps.
  • After evaporation dichloromethane and toluene (1:2, dry 3 ml/g mPEG) was added to re-dissolve the white solid. N-Hydroxy succinimide (2 mole/mole mPEG) was added as a solid and then triethylamine (1.1 mole/mole mPEG). The mixture was stirred for 3 hours, initially unclear, then clear and ending with a small precipitate. The mixture was evaporated to dryness and recrystallized from ethyl acetate (10 ml) with warm filtration to remove salts and insoluble traces. The blank liquid was left for slow cooling at ambient temperature for 16 hours and then in the refrigerator overnight. The white precipitate was filtered and washed with a little cold ethyl acetate and dried to yield 98% (w/w). NMR Indicating 80-90% activation and 5 o/oo (w/w) HNEt3Cl. 1H-NMR for mPEG 15,000 (CDCl3) d 1.42 t (I=4.8 CH3 i HNEt3Cl), 2.84 s (I=3.7 succinimide), 3.10 dq (I=3.4 CH2 i HNEt3Cl), 3.38 s (I=2.7 CH3 i OMe), 3.40* dd (I=4.5 o/oo, 13C satellite), 3.64 bs (I=1364 main peak), 3.89* dd (I=4.8 o/oo, 13C satellite), 4.47 dd (I=1.8, CH2 in PEG). No change was seen after storage in a desiccator at 22° C. for 4 months.
    • Example 6
  • Activation of mPEG 5,000 with N-succinimidyl Carbonate
  • Activation of mPEG 5,000 with N-succinimidyl carbonate was performed as described in Example 5.
    • Example 7
  • Construction and Expression of PD498 Variants:
  • PD498 site-directed variants were constructed using the “maxi-oligonucleotide-PCR” method described by Sarkar et al., 1990, BioTechniques, 8, 404-407.
  • The template plasmid was shuttle vector pPD498 or an analogue of this containing a variant of the PD498 protease gene.
  • The following PD498 variants were constructed, expressed and purified.
    • A: R28K
    • B: R62K
    • C: R169K
    • D: R28K+ R62K
    • E: R28K+ R169K
    • F: R62K+ R169K
    • G: R28K+ R69K+ R169K.
      Construction of Variants
  • For introduction of the R28K substitution a synthetic oligonucleotide having the sequence: GGG ATG TAA CCA AGG GAA GCA GCA CTC AAA CG (SEQ ID NO: 7) was used.
  • A PCR fragment of 769 bp was ligated into the pPD498 plasmid prepared by Bst E II and Bgl II digestion. Positive variants were recognized by Styl digestion and verified by DNA sequencing of the total 769 bp insert.
  • For introduction of the R62K substitution a synthetic oligonucleotide having the sequence: CGA CTT TAT CGA TAA GGA CAA TAA CCC (SEQ ID NO: 8) was used.
  • A PCR fragment of 769 bp was ligated into the pPD498 plasmid prepared by Bst E II and Bgl II digestion. Positive variants were recognized by ClaI digestion and verified by DNA sequencing of the total 769 bp insert.
  • For introduction of the R169K substitution a synthetic oligonucleotide having the sequence: CAA TGT ATC CAA AAC GTT CCA ACC AGC (SEQ ID NO: 9) was used.
  • A PCR fragment of 769 bp was ligated into the pPD498 plasmid prepared by Bst E II and Bgl II digestion. Positive variants were recognized by the absence of an Rsa I restriction site and verified by DNA sequencing of the total 769 bp insert.
  • For simultaneous introduction of the R28K and the R62K substitutions, synthetic oligonucleotides having the sequence GGG ATG TAA CCA AGG GAA GCA GCA CTC AAA CG (SEQ ID NO: 7) and the sequence CGA CTT TAT CGA TAA GGA CAA TAA CCC (SEQ ID NO: 8) were used simultaneously. A PCR fragment of 769 bp was ligated into the pPD498 plasmid prepared by Bst E II and Bgl II digestion. Positive variants were recognized by Styl and ClaI digestion and verified by DNA sequencing of the total 769 bp insert.
  • For simultaneous introduction of the R28K and the R169K substitutions, synthetic oligonucleotides having the sequence GGG ATG TAA CCA AGG GAA GCA GCA CTC AAA CG (SEQ ID NO: 7) and the sequence CAA TGT ATC CAA AAC GTT CCA ACC AGC (SEQ ID NO: 9) were used simultaneously. A PCR fragment of 769 bp was ligated into the pPD498 plasmid prepared by Bst E II and Bgl II digestion. Positive variants were recognized by Styl digestion and absence of an Rsa I site. The variant was verified by DNA sequencing of the total 769 bp insert.
  • For simultaneous ntroduction of the R62K and the R169K substitutions, synthetic oligonucleotides having the sequence CGA CTT TAT CGA TAA GGA CAA TAA CCC (SEQ ID NO: 8) and the sequence CAA TGT ATC CAA AAC GTT CCA ACC AGC (SEQ ID NO: 9) were used simultaneously. A PCR fragment of 769 bp was ligated into the pPD498 plasmid prepared by Bst E II and Bgl II digestion. Positive variants were recognized by ClaI digestion and absence of an Rsa I site. The variant was verified by DNA sequencing of the total 769 bp insert.
  • For simultaneous introduction of the R28K, the R62K and the R169K substitutions, synthetic oligonucleotides having the sequence GGG ATG TAA CCA AGG GAA GCA GCA CTC AAA CG (SEQ ID NO: 7), the sequence CGA CTT TAT CGA TAA GGA CAA TAA CCC (SEQ ID NO: 8) and the sequence CAA TGT ATC CAA AAC GTT CCA ACC AGC (SEQ ID NO: 9) were used simultaneously. A PCR fragment of 769 bp was ligated into the pPD498 plasmid prepared by Bst E II and Bgl II digestion. Positive variants were recognized by Styl and ClaI digestion and absence of an Rsa I site. The variant was verified by DNA sequencing of the total 769 bp insert.
  • Fermentation, Expression and Purification of PD498 Variants
  • Vectors hosting the above mentioned PD498 variants were purified from E. coli cultures and transformed into B. subtilis in which organism the variants were fermented, expressed and purified as described in the “Materials and Methods” section above.
    • Example 8
  • Conjugation of Triple Substituted PD498 Variant with Activated mPEG 5,000
  • 200 mg of triple substituted PD498 variant (i.e. the R28K+ R62K+ R169K substituted variant) was incubated in 50 mm NaBorate, pH 10, with 1.8 g of activated mPEG 5,000 with N-succinimidyl carbonate (prepared according to Example 2), in a final volume of 20 ml. The reaction was carried out at ambient temperature using magnetic stirring. Reaction time was 1 hour. The reaction was stopped by adding DMG buffer to a final concentration of 5 mM dimethyl glutarate, 1 mM CaCl2 and 50 mM borate, pH 5.0.
  • The molecule weight of the obtained derivative was approximately 120 kDa, corresponding to about 16 moles of mPEG attached per mole enzyme.
  • Compared to the parent enzyme, residual activity was close to 100% towards peptide substrate (succinyl-Ala-Ala-Pro-Phe-p-Nitroanilide).
    • Example 9
  • Allergenicity Trials of PD498 Variant-SPEG 5,000 in Guinea Pigs
  • Dunkin Hartley guinea pigs are stimulated with 1.0 microgram PD498-SPEG 5,000 and 1.0 microgram modified variant PD498-SPEG 5,000 by intratracheal installation.
  • Sera from immunized Dunkin Hartley guinea pigs are tested during the trial period in a specific IgG1 ELISA (described above) to elucidate whether the molecules could activate the immune response system giving rise to a specific IgG1 response indicating an allergenic response.
  • The IgG1 levels of Dunkin Hartley guinea pigs during the trial period of 10 weeks are observed.
    • Example 10
  • Suitable Substitutions in Humicola lanuginosa Lipase for Addition of Amino Attachment Groups (—NH2)
  • The 3D structure of Humicola lanuginosa lipase (SEQ ID NO: 6) is available in Brookhaven Databank as ltib.pdb. The lipase consists of 269 amino acids.
  • The procedure described in Example 1 was followed. The sequence of H. lanuginosa lipase is shown below in the table listing solvent accessibility data for H. lanuginosa lipase. H. lanuginosa residue numbering is used (1-269), and the active site residues (functional site) are S146, S201 and H258. The synonym TIB is used for H. lanuginosa lipase.
  • The commands performed in Insight (BIOSYM) are shown in the command files makeKzone.bcl and makeKzone2.bcl below:
  • Conservative Substitutions:
    • makeKzone.bcl
    • 1 Delete Subset *
    • 2 Color Molecule Atoms * Specified Specification 255,0,255
    • 3 Zone Subset LYS :lys:NZ Static monomer/residue 10 Color_Subset 255,255,0
    • 4 Zone Subset NTERM :1:N Static monomer/residue 10 Color_Subset 255,255,0
    • 5 #NOTE: editnextline ACTSITE residues according to the protein
    • 6 Zone Subset ACTSITE :146,201,258 Static monomer/residue 8 Color_Subset 255,255,0
    • 7 Combine Subset ALLZONE Union LYS NTERM
    • 8 Combine Subset ALLZONE Union ALLZONE ACTSITE
    • 9 #NOTE: editnextline object name according to the protein
    • 10 Combine Subset REST Difference TIB ALLZONE
    • 11 List Subset REST Atom Output_File restatom.list
    • 12 List Subset REST monomer/residue Output_File restmole.list
    • 13 Color Molecule Atoms ACTSITE Specified Specification 255,0,0
    • 14 List Subset ACTSITE Atom Output_File actsiteatom.list
    • 15 List Subset ACTSITE monomer/residue Output_File actsitemole.list
    • 16 #
    • 17 Zone Subset REST5A REST Static Monomer/Residue 5-Color_Subset
    • 18 Combine Subset SUB5A Difference REST5A ACTSITE
    • 19 Combine Subset SUB5B Difference SUB5A REST
    • 20 Color Molecule Atoms SUB5B Specified Specification 255,255,255
    • 21 List Subset SUB5B Atom Output_File sub5batom.list
    • 22 List Subset SUB5B monomer/residue Output_File sub5bmole.list
    • 23 #Now identify sites for lys->arg substitutions and continue with makezone2.bcl
    • 24 #Use grep command to identify ARG in restatom.list, sub5batom.list & accsiteatom.list.
      Comments:
  • In this case of H. lanuginosa (=TIB), REST contains the arginines Arg133, Arg139, Arg160, Arg179 and Arg 209, and SUB5B contains Arg118 and R125.
  • These residues are all solvent exposed. The substitutions R133K, R139K, R160K, R179K, R209K, R118K and R125K are identified in TIB as sites for mutagenesis within the scope of this invention. The residues are substituted below in section 2, and further analysis done. The subset ACTSITE contains no lysines.
  • Non-Conservative Substitutions:
    • makeKzone2.bcl
    • 1 #sourcefile makezone2.bcl Claus von der Osten 961128
    • 2 #
    • 3 #having scanned lists (grep arg command) and identified sites for lys->arg substitutions
    • 4 #NOTE: editnextline object name according to protein
    • 5 Copy Object -To_Clipboard -Displace TIB newmodel
    • 6 Biopolymer
    • 7 #NOTE: editnextline object name according to protein
    • 8 Blank Object On TIB
    • 9 #NOTE: editnextlines with lys->arg positions
    • 10 Replace Residue newmodel:118 lys L
    • 11 Replace Residue newmodel:125 lys L
    • 12 Replace Residue newmodel:133 lys L
    • 13 Replace Residue newmodel:139 lys L
    • 14 Replace Residue newmodel:160 lys L
    • 15 Replace Residue newmodel:179 lys L
    • 16 Replace Residue newmodel:209 lys L
    • 17 #
    • 18 #Now repeat analysis done prior to arg->lys, now including introduced lysines
    • 19 Color Molecule Atoms newmodel Specified Specification 255,0,255
    • 20 Zone Subset LYSx newmodel:lys:NZ Static monomer/residue 10 Color_Subset 255,255,0
    • 21 Zone Subset NTERMx newmodel:l:N Static monomer/residue 10 Color_Subset 255,255,0
    • 22 #NOTE: editnextline ACTSITEx residues according to the protein
    • 23 Zone Subset ACTSITEx newmodel:146,201,258 Static monomer/residue 8 Color_Subset 255,255,0
    • 24 Combine Subset ALLZONEx Union LYSx NTERMx
    • 25 Combine Subset ALLZONEx Union ALLZONEx ACTSITEx
    • 26 Combine Subset RESTx Difference newmodel ALLZONEx
    • 27 List Subset RESTx Atom Output_File restxatom.list
    • 28 List Subset RESTx monomer/residue Output_File restxmole.list
    • 29 #
    • 30 Color Molecule Atoms ACTSITEx Specified Specification 255,0,0
    • 31 List Subset ACTSITEx Atom Output_File actsitexatom.list
    • 32 List Subset ACTSITEx monomer/residue Output_File actsitexmole.list
    • 33 #
    • 34 #read restxatom.list or restxmole.list to identify sites for (not_arg)->lys subst. if needed.
      Comments:
  • Of the residues in RESTx, the following are >5% exposed (see lists below): 18, 31-33, 36, 38, 40, 48, 50, 56-62, 64, 78, 88, 91-93, 104-106, 120, 136, 225, 227-229, 250, 262, 268. Of these three are cysteines involved in disulfide bridge formation, and consequently for structural reasons excluded from the residues to be mutated. The following mutations are proposed in H. lanuginosa lipase (TIB): A18K, G31K, T32K, N33K, G38K, A40K, D48K, T50K, E56K, D57K, S58K, G59K, V60K, G61K, D62K, T64K, L78K, N88K, G91K, N92K, L93K, S105K, G106K, V120K, P136K, G225K, L227K, V228K, P229K, P250K, F262K.
  • Relevant Data for Example 10:
    # TIBNOH2O
    # residue area
    GLU_1 110.792610
    VAL_2 18.002457
    SER_3 53.019516
    GLN_4 85.770164
    ASP_5 107.565826
    LEU_6 33.022659
    PHE_7 34.392754
    ASN_8 84.855331
    GLN_9 39.175591
    PHE_10 2.149547
    ASN_11 40.544380
    LEU_12 27.648788
    PHE_13 2.418241
    ALA_14 4.625293
    GLN_15 28.202387
    TYR_16 0.969180
    SER_17 0.000000
    ALA_18 7.008336
    ALA_19 0.000000
    ALA_20 0.000000
    TYR_21 6.947358
    CYS_22 8.060802
    GLY_23 32.147034
    LYS_24 168.890747
    ASN_25 8.014721
    ASN_26 11.815564
    ASP_27 92.263428
    ALA_28 18.206699
    PRO_29 83.188431
    ALA_30 69.428421
    GLY_31 50.693439
    THR_32 52.171135
    ASN_33 111.230743
    ILE_34 2.801945
    THR_35 82.130569
    CYS_36 17.269245
    THR_37 96.731941
    GLY_38 77.870995
    ASN_39 123.051003
    ALA_40 27.985256
    CYS_41 0.752820
    PRO_42 46.258949
    GLU_43 69.773987
    VAL_44 0.735684
    GLU_45 77.169510
    LYS_46 141.213562
    ALA_47 10.249716
    ASP_48 109.913902
    ALA_49 2.602721
    THR_50 32.012184
    PHE_51 8.255627
    LEU_52 60.093613
    TYR_53 77.877937
    SER_54 26.980494
    PHE_55 10.747735
    GLU_56 112.689758
    ASP_57 92.064278
    SER_58 32.990780
    GLY_59 53.371807
    VAL_60 83.563644
    GLY_61 69.625633
    ASP_62 75.520988
    VAL_63 4.030401
    THR_64 8.652839
    GLY_65 0.000000
    PHE_66 0.268693
    LEU_67 11.822510
    ALA_68 0.537387
    LEU_69 30.243870
    ASP_70 0.000000
    ASN_71 84.101044
    THR_72 89.271126
    ASN_73 70.742401
    LYS_74 98.319168
    LEU_75 8.329495
    ILE_76 5.197878
    VAL_77 0.806080
    LEU_78 5.293978
    SER_79 0.000000
    PHE_80 2.079151
    ARG_81 41.085312
    GLY_82 1.471369
    SER_83 43.794014
    ARG_84 100.261627
    SER_85 70.607552
    ILE_86 59.696865
    GLU_87 136.510773
    ASN_88 119.376373
    TRP_89 102.851227
    ILE_90 78.068588
    GLY_91 60.783607
    ASN_92 45.769428
    LEU_93 134.228363
    ASN_94 101.810959
    PHE_95 41.212212
    ASP_96 79.645950
    LEU_97 25.281572
    LYS_98 88.840263
    GLU_99 132.377090
    ILE_100 9.135575
    ASN_101 63.444527
    ASP_102 88.652847
    ILE_103 33.470661
    CYS_104 11.553816
    SER_105 99.461174
    GLY_106 40.325161
    CYS_107 4.433561
    ARG_108 97.450104
    GLY_109 1.343467
    HIS_110 4.652464
    ASP_111 37.023655
    GLY_112 29.930408
    PHE_113 14.976435
    THR_114 10.430954
    SER_115 40.606895
    SER_116 13.462922
    TRP_117 10.747735
    ARG_118 114.364281
    SER_119 46.880249
    VAL_120 13.434669
    ALA_121 18.258261
    ASP_122 110.753098
    THR_123 69.641922
    LEU_124 17.090784
    ARG_125 73.929977
    GLN_126 101.320190
    LYS_127 84.450241
    VAL_128 6.448641
    GLU_129 47.700993
    ASP_130 75.529091
    ALA_131 11.340775
    VAL_132 27.896025
    ARG_133 153.136490
    GLU_134 132.140594
    HIS_135 54.553406
    PRO_136 97.386963
    ASP_137 22.653191
    TYR_138 35.392658
    ARG_139 74.321243
    VAL_140 10.173222
    VAL_141 0.233495
    PHE_142 3.224321
    THR_143 0.000000
    GLY_144 0.000000
    HIS_145 4.514527
    SER_146 15.749787
    LEU_147 40.709171
    GLY_148 0.000000
    GLY_149 0.000000
    ALA_150 0.537387
    LEU_151 22.838938
    ALA_152 0.268693
    THR_153 18.078798
    VAL_154 7.254722
    ALA_155 0.000000
    GLY_156 0.000000
    ALA_157 15.140230
    ASP_158 41.645477
    LEU_159 6.144750
    ARG_160 41.939716
    GLY_161 68.978180
    ASN_162 68.243805
    GLY_163 79.181274
    TYR_164 36.190247
    ASP_165 103.068283
    ILE_166 0.000000
    ASP_167 24.326443
    VAL_168 4.299094
    PHE_169 0.466991
    SER_170 3.339332
    TYR_171 0.000000
    GLY_172 0.000000
    ALA_173 12.674671
    PRO_174 13.117888
    ARG_175 10.004488
    VAL_176 21.422220
    GLY_177 2.680759
    ASN_178 21.018063
    ARG_179 110.282166
    ALA_180 33.210381
    PHE_181 4.567788
    ALA_182 3.897251
    GLU_183 76.354004
    PHE_184 71.225983
    LEU_185 24.985012
    THR_186 47.023815
    VAL_187 98.244606
    GLN_188 54.152954
    THR_189 88.660645
    GLY_190 24.792120
    GLY_191 10.726818
    THR_192 45.458744
    LEU_193 16.633211
    TYR_194 34.829491
    ARG_195 29.030851
    ILE_196 1.973557
    THR_197 3.493014
    HIS_198 1.532270
    THR_199 34.785877
    ASN_200 39.789238
    ASP_201 0.000000
    ILE_202 31.168434
    VAL_203 29.521076
    PRO_204 3.515322
    ARG_205 44.882454
    LEU_206 51.051746
    PRO_207 12.575329
    PRO_208 43.259636
    ARG_209 113.700233
    GLU_210 154.628540
    PHE_211 112.505188
    GLY_212 30.084938
    TYR_213 3.268936
    SER_214 12.471436
    HIS_215 23.354481
    SER_216 16.406200
    SER_217 14.665598
    PRO_218 17.240993
    GLU_219 13.145291
    TYR_220 18.718306
    TRP_221 39.229233
    ILE_222 5.105175
    LYS_223 120.739983
    SER_224 15.407301
    GLY_225 29.306646
    THR_226 66.806862
    LEU_227 122.682808
    VAL_228 60.923004
    PRO_229 104.620377
    VAL_230 23.398251
    THR_231 63.372971
    ARG_232 80.357857
    ASN_233 89.255066
    ASP_234 43.011250
    ILE_235 2.114349
    VAL_236 45.140491
    LYS_237 105.651306
    ILE_238 24.671705
    GLU_239 116.891907
    GLY_240 31.965794
    ILE_241 46.278099
    ASP_242 28.963699
    ALA_243 25.158146
    THR_244 98.351440
    GLY_245 43.842186
    GLY_246 0.700486
    ASN_247 3.926274
    ASN_248 51.047890
    GLN_249 66.699188
    PRO_250 132.414047
    ASN_251 70.213730
    ILE_252 141.498062
    PRO_253 59.089233
    ASP_254 59.010895
    ILE_255 63.298943
    PRO_256 78.608688
    ALA_257 0.806080
    HIS_258 3.761708
    LEU_259 50.747856
    TRP_260 35.229710
    TYR_261 5.440791
    PHE_262 36.457939
    GLY_263 22.071375
    LEU_264 109.148178
    ILE_265 2.418241
    GLY_266 17.730062
    THR_267 68.217873
    CYS_268 15.418195
    LEU_269 165.990997
    Subset REST:
    restmole.list
    Subset REST:
    TIB: 5, 8-9, 13-14, 16, 18-20, 31-34, 36, 38, 40, 48-50, 56-66, 68, 76-79, 88,
    91-93,
    TIB: 100-107, 116-117, 119-121, 132-134, 136, 139-142, 154-169, 177-185,
    TIB: 187, 189-191, 207-212, 214-216, 225, 227-229, 241-244, 250, 262, 268
    restatom.list
    Subset REST:
    TIB: ASP 5: N, CA, C, O, CB, CG, OD1, OD2
    TIB: ASN 8: N, CA, C, O, CB, CG, OD1, ND2
    TIB: GLN 9: N, CA, C, O, CB, CG, CD, OE1, NE2
    TIB: PHE 13: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ
    TIB: ALA 14: N, CA, C, O, CB
    TIB: TYR 16: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ, OH
    TIB: ALA 18: N, CA, C, O, CB
    TIB: ALA 19: N, CA, C, O, CB
    TIB: ALA 20: N, CA, C, O, CB
    TIB: GLY 31: N, CA, C, O
    TIB: THR 32: N, CA, C, O, CB, OG1, CG2
    TIB: ASN 33: N, CA, C, O, CB, CG, OD1, ND2
    TIB: ILE 34: N, CA, C, O, CB, CG1, CG2, CD1
    TIB: CYS 36: N, CA, C, O, CB, SG
    TIB: GLY 38: N, CA, C, O
    TIB: ALA 40: N, CA, C, O, CB
    TIB: ASP 48: N, CA, C, O, CB, CG, OD1, OD2
    TIB: ALA 49: N, CA, C, O, CB
    TIB: THR 50: N, CA, C, O, CB, OG1, CG2
    TIB: GLU 56: N, CA, C, O, CB, CG, CD, OE1, OE2
    TIB: ASP 57: N, CA, C, O, CB, CG, OD1, OD2
    TIB: SER 58: N, CA, C, O, CB, OG
    TIB: GLY 59: N, CA, C, O
    TIB: VAL 60: N, CA, C, O, CB, CG1, CG2
    TIB: GLY 61: N, CA, C, O
    TIB: ASP 62: N, CA, C, O, CB, CG, OD1, OD2
    TIB: VAL 63: N, CA, C, O, CB, CG1, CG2
    TIB: THR 64: N, CA, C, O, CB, OG1, CG2
    TIB: GLY 65: N, CA, C, O
    TIB: PHE 66: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ
    TIB: ALA 68: N, CA, C, O, CB
    TIB: ILE 76: N, CA, C, O, CB, CG1, CG2, CD1
    TIB: VAL 77: N, CA, C, O, CB, CG1, CG2
    TIB: LEU 78: N, CA, C, O, CB, CG, CD1, CD2
    TIB: SER 79: N, CA, C, O, CB, OG
    TIB: ASN 88: N, CA, C, O, CB, CG, OD1, ND2
    TIB: GLY 91: N, CA, C, O
    TIB: ASN 92: N, CA, C, O, CB, CG, OD1, ND2
    TIB: LEU 93: N, CA, C, O, CB, CG, CD1, CD2
    TIB: ILE 100: N, CA, C, O, CB, CG1, CG2, CD1
    TIB: ASN 101: N, CA, C, O, CB, CG, OD1, ND2
    TIB: ASP 102: N, CA, C, O, CB, CG, OD1, OD2
    TIB: ILE 103: N, CA, C, O, CB, CG1, CG2, CD1
    TIB: CYS 104: N, CA, C, O, CB, SG
    TIB: SER 105: N, CA, C, O, CB, OG
    TIB: GLY 106: N, CA, C, O
    TIB: CYS 107: N, CA, C, O, CB, SG
    TIB: SER 116: N, CA, C, O, CB, OG
    TIB: TRP 117: N, CA, C, O, CB, CG, CD1, CD2, NE1, CE2, CE3, CZ2, CZ3, CH2
    TIB: SER 119: N, CA, C, O, CB, OG
    TIB: VAL 120: N, CA, C, O, CB, CG1, CG2
    TIB: ALA 121: N, CA, C, O, CB
    TIB: VAL 132: N, CA, C, O, CB, CG1, CG2
    TIB: ARG 133: N, CA, C, O, CB, CG, CD, NE, CZ, NH1, NH2
    TIB: GLU 134: N, CA, C, O, CB, CG, CD, OE1, OE2
    TIB: PRO 136: N, CA, CD, C, O, CB, CG
    TIB: ARG 139: N, CA, C, O, CB, CG, CD, NE, CZ, NH1, NH2
    TIB: VAL 140: N, CA, C, O, CB, CG1, CG2
    TIB: VAL 141: N, CA, C, O, CB, CG1, CG2
    TIB: PHE 142: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ
    TIB: VAL 154: N, CA, C, O, CB, CG1, CG2
    TIB: ALA 155: N, CA, C, O, CB
    TIB: GLY 156: N, CA, C, O
    TIB: ALA 157: N, CA, C, O, CB
    TIB: ASP 158: N, CA, C, O, CB, CG, OD1, OD2
    TIB: LEU 159: N, CA, C, O, CB, CG, CD1, CD2
    TIB: ARG 160: N, CA, C, O, CB, CG, CD, NE, CZ, NH1, NH2
    TIB: GLY 161: N, CA, C, O
    TIB: ASN 162: N, CA, C, O, CB, CG, OD1, ND2
    TIB: GLY 163: N, CA, C, O
    TIB: TYR 164: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ, OH
    TIB: ASP 165: N, CA, C, O, CB, CG, OD1, OD2
    TIB: ILE 166: N, CA, C, O, CB, CG1, CG2, CD1
    TIB: ASP 167: N, CA, C, O, CB, CG, OD1, OD2
    TIB: VAL 168: N, CA, C, O, CB, CG1, CG2
    TIB: PHE 169: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ
    TIB: GLY 177: N, CA, C, O
    TIB: ASN 178: N, CA, C, O, CB, CG, CD1, ND2
    TIB: ARG 179: N, CA, C, O, CB, CG, CD, NE, CZ, NH1, NH2
    TIB: ALA 180: N, CA, C, O, CB
    TIB: PHE 181: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ
    TIB: ALA 182: N, CA, C, O, CB
    TIB: GLU 183: N, CA, C, O, CB, CG, CD, OE1, OE2
    TIB: PHE 184: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ
    TIB: LEU 185: N, CA, C, O, CB, CG, CD1, CD2
    TIB: VAL 187: N, CA, C, O, CB, CG1, CG2
    TIB: THR 189: N, CA, C, O, CB, OG1, CG2
    TIB: GLY 190: N, CA, C, O
    TIB: GLY 191: N, CA, C, O
    TIB: PRO 207: N, CA, CD, C, O, CB, CG
    TIB: PRO 208: N, CA, CD, C, O, CB, CG
    TIB: ARG 209: N, CA, C, O, CB, CG, CD, NE, CZ, NH1, NH2
    TIB: GLU 210: N, CA, C, O, CB, CG, CD, OE1, OE2
    TIB: PHE 211: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ
    TIB: GLY 212: N, CA, C, O
    TIB: SER 214: N, CA, C, O, CB, OG
    TIB: HIS 215: N, CA, C, O, CB, CG, ND1, CD2, CE1, ME2
    TIB: SER 216: N, CA, C, O, CB, OG
    TIB: GLY 225: N, CA, C, O
    TIB: LEU 227: N, CA, C, O, CB, CG, CD1, CD2
    TIB: VAL 228: N, CA, C, O, CB, CG1, CG2
    TIB: PRO 229: N, CA, CD, C, O, CB, CG
    TIB: ILE 241: N, CA, C, O, CB, CG1, CG2, CD1
    TIB: ASP 242: N, CA, C, O, CB, CG, OD1, OD2
    TIB: ALA 243: N, CA, C, O, CB
    TIB: THR 244: N, CA, C, O, CB, OG1, CG2
    TIB: PRO 250: N, CA, CD, C, O, CB, CG
    TIB: PHE 262: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ
    TIB: CYS 268: N, CA, C, O, CB, SG
    Subset SUB5B:
    sub5mole.list
    Subset SUB5B:
    TIB: 3-4, 6-7, 10-12, 15, 22-23, 25-30, 35, 37, 39, 41-42, 44-47, 51-55, 67,
    69-70,
    TIB: 72, 74-75, 94-99, 108-112, 114-115, 118, 122-126, 128-131, 135,
    137-138,
    TIB: 186, 188, 192-195, 213, 217-219, 223-224, 230-231, 234-235, 238-240,
    TIB: 245, 269
    sub5batom.list
    Subset SUB5B:
    TIB: SER 3: N, CA, C, O, CB, OG
    TIB: GLN 4: N, CA, C, O, CB, CG, CD, OE1, NE2
    TIB: LEU 6: N, CA, C, O, CB, CG, CD1, CD2
    TIB: PHE 7: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ
    TIB: PHE 10: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ
    TIB: ASN 11: N, CA, C, O, CB, CG, OD1, ND2
    TIB: LEU 12: N, CA, C, O, CB, CG, CD1, CD2
    TIB: GLN 15: N, CA, C, O, CB, CG, CD, OE1, NE2
    TIB: CYS 22: N, CA, C, O, CB, SG
    TIB: GLY 23: N, CA, C, O
    TIB: ASN 25: N, CA, C, O, CB, CG, OD1, ND2
    TIB: ASN 26: N, CA, C, O, CB, CG, OD1, ND2
    TIB: ASP 27: N, CA, C, O, CB, CG, OD1, OD2
    TIB: ALA 28: N, CA, C, O, CB
    TIB: PRO 29: N, CA, CD, C, O, CB, CG
    TIB: ALA 30: N, CA, C, O, CB
    TIB: THR 35: N, CA, C, O, CB, OG1, CG2
    TIB: THR 37: N, CA, C, O, CB, OG1, CG2
    TIB: ASN 39: N, CA, C, O, CB, CG, OD1, ND2
    TIB: CYS 41: N, CA, C, O, CB, SG
    TIB: PRO 42: N, CA, CD, C, O, CB, CG
    TIB: VAL 44: N, CA, C, O, CB, CG1, CG2
    TIB: GLU 45: N, CA, C, O, CB, CG, CD, OE1, OE2
    TIB: LYS 46: N, CA, C, O, CB, CG, CD, CE, NZ
    TIB: ALA 47: N, CA, C, O, CB
    TIB: PHE 51: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ
    TIB: LEU 52: N, CA, C, O, CB, CG, CD1, CD2
    TIB: TYR 53: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ, OH
    TIB: SER 54: N, CA, C, O, CB, OG
    TIB: PHE 55: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ
    TIB: LEU 67: N, CA, C, O, CB, CG, CD1, CD2
    TIB: LEU 69: N, CA, C, O, CB, CG, CD1, CD2
    TIB: ASP 70: N, CA, C, O, CB, CG, OD1, OD2
    TIB: THR 72: N, CA, C, O, CB, OG1, CG2
    TIB: LYS 74: N, CA, C, O, CB, CG, CD, CE, NZ
    TIB: LEU 75: N, CA, C, O, CB, CG, CD1, CD2
    TIB: ASN 94: N, CA, C, O, CB, CG, OD1, ND2
    TIB: PHE 95: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ
    TIB: ASP 96: N, CA, C, O, CB, CG, OD1, OD2
    TIB: LEU 97: N, CA, C, O, CB, CG, CD1, CD2
    TIB: LYS 98: N, CA, C, O, CB, CG, CD, CE, NZ
    TIB: GLU 99: N, CA, C, O, CB, CG, CD, OE1, OE2
    TIB: ARG 108: N, CA, C, O, CB, CG, CD, NE, CZ, NH1, NH2
    TIB: GLY 109: N, CA, C, O
    TIB: HIS 110: N, CA, C, O, CB, CG, ND1, CD2, CE1, NE2
    TIB: ASP 111: N, CA, C, O, CB, CG, OD1, OD2
    TIB: GLY 112: N, CA, C, O
    TIB: THR 114: N, CA, C, O, CB, OG1, CG2
    TIB: SER 115: N, CA, C, O, CB, OG
    TIB: ARG 118: N, CA, C, O, CB, CG, CD, NE, CZ, NH1, NH2
    TIB: ASP 122: N, CA, C, O, CB, CG, OD1, OD2
    TIB: THR 123: N, CA, C, O, CB, OG1, CG2
    TIB: LEU 124: N, CA, C, O, CB, CG, CD1, CD2
    TIB: ARG 125: N, CA, C, O, CB, CG, CD, NE, CZ, NH1, NH2
    TIB: GLN 126: N, CA, C, O, CB, CG, CD, OE1, NE2
    TIB: VAL 128: N, CA, C, O, CB, CG1, CG2
    TIB: GLU 129: N, CA, C, O, CB, CG, CD, OE1, OE2
    TIB: ASP 130: N, CA, C, O, CB, CG, OD1, OD2
    TIB: ALA 131: N, CA, C, O, CB
    TIB: HIS 135: N, CA, C, O, CB, CG, ND1, CD2, CE1, NE2
    TIB: ASP 137: N, CA, C, O, CB, CG, OD1, OD2
    TIB: TYR 138: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ, OH
    TIB: THR 186: N, CA, C, O, CB, OG1, CG2
    TIB: GLN 188: N, CA, C, O, CB, CG, CD, OE1, NE2
    TIB: THR 192: N, CA, C, O, CB, OG1, CG2
    TIB: LEU 193: N, CA, C, O, CB, CG, CD1, CD2
    TIB: TYR 194: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ, OH
    TIB: ARG 195: N, CA, C, O, CB, CG, CD, NE, CZ, NH1, NH2
    TIB: TYR 213: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ, OH
    TIB: SER 217: N, CA, C, O, CB, OG
    TIB: PRO 218: N, CA, CD, C, O, CB, CG
    TIB: GLU 219: N, CA, C, O, CB, CG, CD, OE1, OE2
    TIB: LYS 223: N, CA, C, O, CB, CG, CD, CE, NZ
    TIB: SER 224: N, CA, C, O, CB, OG
    TIB: VAL 230: N, CA, C, O, CB, CG1, CG2
    TIB: THR 231: N, CA, C, O, CB, OG1, CG2
    TIB: ASP 234: N, CA, C, O, CB, CG, OD1, OD2
    TIB: ILE 235: N, CA, C, O, CB, CG1, CG2, CD1
    TIB: ILE 238: N, CA, C, O, CB, CG1, CG2, CD1
    TIB: GLU 239: N, CA, C, O, CB, CG, CD, OE1, OE2
    TIB: GLY 240: N, CA, C, O
    TIB: GLY 245: N, CA, C, O
    TIB: LEU 269: N, CA, C, O, CB, OXT, CG, CD1, CD2
    Subset ACTSITE:
    actsitemole.list
    Subset ACTSITE:
    TIB: 17, 21, 80-87, 89-90, 113, 143-153, 170-176, 196-206, 221-222, 226,
    246-249,
    TIB: 251-261, 263-267
    actsiteatom.list
    Subset ACTSITE:
    TIB: SER 17: N, CA, C, O, CB, OG
    TIB: TYR 21: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ, OH
    TIB: PHE 80: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ
    TIB: ARG 81: N, CA, C, O, CB, CG, CD, NE, CZ, NH1, NH2
    TIB: GLY 82: N, CA, C, O
    TIB: SER 83: N, CA, C, O, CB, OG
    TIB: ARG 84: N, CA, C, O, CB, CG, CD, NE, CZ, NH1, NH2
    TIB: SER 85: N, CA, C, O, CB, OG
    TIB: ILE 86: N, CA, C, O, CB, CG1, CG2, CD1
    TIB: GLU 87: N, CA, C, O, CB, CG, CD, OE1, OE2
    TIB: TRP 89: N, CA, C, O, CB, CG, CD1, CD2, NE1, CE2, CE3, CZ2, CZ3, CH2
    TIB: ILE 90: N, CA, C, O, CB, CG1, CG2, CD1
    TIB: PHE 113: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ
    TIB: THR 143: N, CA, C, O, CB, OG1, CG2
    TIB: GLY 144: N, CA, C, O
    TIB: HIS 145: N, CA, C, O, CB, CG, ND1, CD2, CE1, NE2
    TIB: SER 146: N, CA, C, O, CB, OG
    TIB: LEU 147: N, CA, C, O, CB, CG, CD1, CD2
    TIB: GLY 148: N, CA, C, O
    TIB: GLY 149: N, CA, C, O
    TIB: ALA 150: N, CA, C, O, CB
    TIB: LEU 151: N, CA, C, O, CB, CG, CD1, CD2
    TIB: ALA 152: N, CA, C, O, CB
    TIB: THR 153: N, CA, C, O, CB, OG1, CG2
    TIB: SER 170: N, CA, C, O, CB, OG
    TIB: TYR 171: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ, OH
    TIB: GLY 172: N, CA, C, O
    TIB: ALA 173: N, CA, C, O, CB
    TIB: PRO 174: N, CA, CD, C, O, CB, CG
    TIB: ARG 175: N, CA, C, O, CB, CG, CD, NE, CZ, NH1, NH2
    TIB: VAL 176: N, CA, C, O, CB, CG1, CG2
    TIB: ILE 196: N, CA, C, O, CB, CG1, CG2, CD1
    TIB: THR 197: N, CA, C, O, CB, OG1, CG2
    TIB: HIS 198: N, CA, C, O, CB, CG, ND1, CD2, CE1, NE2
    TIB: THR 199: N, CA, C, O, CB, OG1, CG2
    TIB: ASN 200: N, CA, C, O, CB, CG, CD1, ND2
    TIB: ASP 201: N, CA, C, O, CB, CG, OD1, OD2
    TIB: ILE 202: N, CA, C, O, CB, CG1, CG2, CD1
    TIB: VAL 203: N, CA, C, O, CB, CG1, CG2
    TIB: PRO 204: N, CA, CD, C, O, CB, CG
    TIB: ARG 205: N, CA, C, O, CB, CG, CD, NE, CZ, NH1, NH2
    TIB: LEU 206: N, CA, C, O, CB, CG, CD1, CD2
    TIB: TRP 221: N, CA, C, O, CB, CG, CD1, CD2, NE1, CE2, CE3, CZ2, CZ3, CH2
    TIB: ILE 222: N, CA, C, O, CB, CG1, CG2, CD1
    TIB: THR 226: N, CA, C, O, CB, OG1, CG2
    TIB: GLY 246: N, CA, C, O
    TIB: ASN 247: N, CA, C, O, CB, CG, OD1, ND2
    TIB: ASN 248: N, CA, C, O, CB, CG, OD1, ND2
    TIB: GLN 249: N, CA, C, O, CB, CG, CD, OE1, NE2
    TIB: ASN 251: N, CA, C, O, CB, CG, OD1, ND2
    TIB: ILE 252: N, CA, C, O, CB, CG1, CG2, CD1
    TIB: PRO 253: N, CA, CD, C, O, CB, CG
    TIB: ASP 254: N, CA, C, O, CB, CG, OD1, OD2
    TIB: ILE 255: N, CA, C, O, CB, CG1, CG2, CD1
    TIB: PRO 256: N, CA, CD, C, O, CB, CG
    TIB: ALA 257: N, CA, C, O, CB
    TIB: HIS 258: N, CA, C, O, CB, CG, ND1, CD2, CE1, NE2
    TIB: LEU 259: N, CA, C, O, CB, CG, CD1, CD2
    TIB: TRP 260: N, CA, C, O, CB, CG, CD1, CD2, NE1, CE2, CE3, CZ2, CZ3, CH2
    TIB: TYR 261: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ, OH
    TIB: GLY 263: N, CA, C, O
    TIB: LEU 264: N, CA, C, O, CB, CG, CD1, CD2
    TIB: ILE 265: N, CA, C, O, CB, CG1, CG2, CD1
    TIB: GLY 266: N, CA, C, O
    TIB: THR 267: N, CA, C, O, CB, OG1, CG2
    Subset RESTX:
    restxmole.list
    Subset RESTX:
    NEWMODEL: 14, 16, 18-20, 31-34, 36, 38, 40, 48-50, 56-66, 68, 78-79, 88, 91-93,
    NEWMODEL: 104-106, 120, 136, 225, 227-229, 250, 262, 268
    restxatom.list
    Subset RESTX:
    NEWMODEL: ALA 14: N, CA, C, O, CB
    NEWMODEL: TYR 16: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ, OH
    NEWMODEL: ALA 18: N, CA, C, O, CB
    NEWMODEL: ALA 19: N, CA, C, O, CB
    NEWMODEL: ALA 20: N, CA, C, O, CB
    NEWMODEL: GLY 31: N, CA, C, O
    NEWMODEL: THR 32: N, CA, C, O, CB, OG1, CG2
    NEWMODEL: ASN 33: N, CA, C, O, CB, CG, OD1, ND2
    NEWMODEL: ILE 34: N, CA, C, O, CB, CG1, CG2, CD1
    NEWMODEL: CYS 36: N, CA, C, O, CB, SG
    NEWMODEL: GLY 38: N, CA, C, O
    NEWMODEL: ALA 40: N, CA, C, O, CB
    NEWMODEL: ASP 48: N, CA, C, O, CB, CG, OD1, OD2
    NEWMODEL: ALA 49: N, CA, C, O, CB
    NEWMODEL: THR 50: N, CA, C, O, CB, OG1, CG2
    NEWMODEL: GLU 56: N, CA, C, O, CB, CG, CD, OE1, OE2
    NEWMODEL: ASP 57: N, CA, C, O, CB, CG, OD1, OD2
    NEWMODEL: SER 58: N, CA, C, O, CB, OG
    NEWMODEL: GLY 59: N, CA, C, O
    NEWMODEL: VAL 60: N, CA, C, O, CB, CG1, CG2
    NEWMODEL: GLY 61: N, CA, C, O
    NEWMODEL: ASP 62: N, CA, C, O, CB, CG, OD1, OD2
    NEWMODEL: VAL 63: N, CA, C, O, CB, CG1, CG2
    NEWMODEL: THR 64: N, CA, C, O, CB, OG1, CG2
    NEWMODEL: GLY 65: N, CA, C, O
    NEWMODEL: PHE 66: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ
    NEWMODEL: ALA 68: N, CA, C, O, CB
    NEWMODEL: LEU 78: N, CA, C, O, CB, CG, CD1, CD2
    NEWMODEL: SER 79: N, CA, C, O, CB, OG
    NEWMODEL: ASN 88: N, CA, C, O, CB, CG, OD1, ND2
    NEWMODEL: GLY 91: N, CA, C, O
    NEWMODEL: ASN 92: N, CA, C, O, CB, CG, OD1, ND2
    NEWMODEL: LEU 93: N, CA, C, O, CB, CG, CD1, CD2
    NEWMODEL: CYS 104: N, CA, C, O, CB, SG
    NEWMODEL: SER 105: N, CA, C, O, CB, OG
    NEWMODEL: GLY 106: N, CA, C, O
    NEWMODEL: VAL 120: N, CA, C, O, CB, CG1, CG2
    NEWMODEL: PRO 136: N, CA, CD, C, O, CB, CG
    NEWMODEL: GLY 225: N, CA, C, O
    NEWMODEL: LEU 227: N, CA, C, O, CB, CG, CD1, CD2
    NEWMODEL: VAL 228: N, CA, C, O, CB, CG1, CG2
    NEWMODEL: PRO 229: N, CA, CD, C, O, CB, CG
    NEWMODEL: PRO 250: N, CA, CD, C, O, CB, CG
    NEWMODEL: PHE 262: N, CA, C, O, CB, CG, CD1, CD2, CE1, CE2, CZ
    NEWMODEL: CYS 268: N, CA, C, O, CB, SG
    • Example 11
  • Providing a Lipase Variant E87K+ D254K
  • The Humicola lanuginosa lipase variant E87K+ D254K was constructed, expressed and purified as described in WO 92/05249.
    • Example 12
  • Lipase-S-PEG 15,000 Conjugate
  • The lipase variant E87K+ D254K-SPEG conjugate was prepared as described in Example 7, except that the enzyme is the Humicola lanuginosa lipase variant (E87K+ D254K) described in Example 11 and the polymer is mPEG 15,000.
    • Example 13
  • Immunogenicity Assessed as IgG1 of Lipase Variant (D87K+ D254K) in Balb/C Mice
  • Balb/c mice were immunized by subcutaneous injection of:
      • (i) 50 microliters 0.9% (wt/vol) NaCl solution (control group, 8 mice) (control),
      • (ii) 50 microliters 0.9% (wt/vol) NaCl solution containing 25 micrograms of protein of a Humicola lanuginosa lipase variant (E87K+ D254K) (group 1, 8 mice) (unmodified lipase variant),
      • (iii) 50% 0.9% (wt/vol) NaCl solution containing a Humicola lanugoinosa lipase variant substituted in position D87K+ D254K and coupled to an N-succinimidyl carbonate activated mPEG 15,000(group 2, 8 mice) (lipase-SPEG 15,000).
  • The amount of protein for each batch was measured by optical density measurements. Blood samples (200 microliters) were collected from the eyes one week after the immunization, but before the following immunization. Serum was obtained by blood clotting, and centrifugation.
  • The IgG1 response was determined by use of the Balb/C mice IgG1 ELISA method as described above.
  • Results:
  • Five weekly immunizations were required to elicit a detectable humoral response to the unmodified Humicola lanuginosa variant. The antibody titers elicited by the conjugate (i.e. lipase-SPEG15,000 ranged between 960 and 1920, and were only 2 to 4× lower than the antibody titer of 3840 that was elicited by unmodified HL82-LIPOLASE (figure to the left).
  • The results of the tests are shown in FIG. 1.
  • As will be apparent to those skilled in the art, in the light of the foregoing disclosure, many alterations and modifications are possible in the practice of this invention without departing from the spirit or scope thereof. Accordingly, the scope of the invention is to be construed in accordance with the substance defined by the following claims.

Claims (15)

1-35. (canceled)
36. A conjugate comprising a lipase moiety conjugated to one or more polymers, wherein the lipase moiety is a Humicola lanuginosa lipase which comprises one or more of the following substitutions:
A18K, G31K, T32K, N33K, G38K, A40K, D48K, T50K, E56K, D57K, S58K, G59K, V60K, G61K, D62K, T64K, L78K, N88K, G91K, N92K, L93K, S105K, G106K, R118K, V120K, R125K, R133K, P136K, R139K, R160K, R179K, R209K, G225K, L227K, V228K, P229K, P250K and F262K.
37. The conjugate of claim 36, wherein the Humicola lanuginosa lipase has an amino acid sequence of SEQ ID NO: 6.
38. The conjugate of claim 36, wherein the polymer(s) have a molecular weight from 1 to 60 kDa.
39. The conjugate of claim 36, wherein the polymer(s) are natural or synthetic homo or heteropolymers.
40. The conjugate of claim 36, wherein the polymer(s) are selected from the group consisting of polyols, polyamines, polycarboxyl acids and polymers comprising a hydroxyl group and an amine group.
41. The conjugate of claim 36, wherein the polymer(s) are selected from the group consisting of polyalkylene oxides (PAO), PEG-glycidyl ethers (Epox-PEG), PEG-oxycarbonylimidazole (CDI-PEG), branched PEGs, polyvinyl alcohols (PVA), poly-carboxylates, polyvinylpyrolidones, poly-D,L-amino acids, polyethylene-co-maleic acid anhydride, polystyrene-co-malic acid anhydrides, dextrans, heparins, homologous albumins, celluloses, hydrolysates of chitosan, starches, glycogen, agaroses and derivatives thereof, guar gum, pullulan, inulin, xanthan gum, carrageenin, pectin, alginic acid hydrolysates and bio-polymers.
42. The conjugate of claim 36, wherein the polymer(s) are polyalkylene glycols (PAG) or methoxypolyethylene glycols (mPEG).
43. The conjugate of claim 36, wherein the polymer(s) are selected from the group consisting of polyethylene glycols (PEG), polypropylene glycols and carboxymethyl-dextrans.
44. The conjugate of claim 36, wherein the polymer(s) are selected from the group consisting of methylcellulose, carboxymethylcellulose, ethylcellulose, hydroxyethylcellulose carboxyethylcellulose and hydroxypropylcellulose.
45. The conjugate of claim 36, wherein the polymer(s) are hydroxyethyl-starches or hydroxypropyl-starches.
46. The conjugate of claim 36, wherein the polymer(s) are methoxypolyethylene glycols (mPEG).
47. A detergent composition comprising a conjugate of claim 36 and a surfactant.
48. A skin care composition, comprising a conjugate of claim 36 and ingredients used in skin care products.
49. A pharmaceutical composition comprising a conjugate of claim 36 and further comprising ingredients used in pharmaceuticals.
US10/623,292 1997-02-06 2003-07-18 Modified enzymes having polymer conjugates Abandoned US20050079593A1 (en)

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DK0135/97 1997-02-06
PCT/DK1998/000046 WO1998035026A1 (en) 1997-02-06 1998-02-06 Polypeptide-polymer conjugates having added and/or removed attachment groups
US09/024,532 US6245901B1 (en) 1997-02-06 1998-02-17 Modified polypeptide
US09/705,185 US6623950B1 (en) 1997-02-06 2000-11-02 Modified enzymes having polymer conjugates
US10/623,292 US20050079593A1 (en) 1997-02-06 2003-07-18 Modified enzymes having polymer conjugates

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