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WO1995001425A1 - Compositions renfermant des agents oxydo-reducteurs de bisulphure proteique - Google Patents

Compositions renfermant des agents oxydo-reducteurs de bisulphure proteique Download PDF

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Publication number
WO1995001425A1
WO1995001425A1 PCT/DK1994/000265 DK9400265W WO9501425A1 WO 1995001425 A1 WO1995001425 A1 WO 1995001425A1 DK 9400265 W DK9400265 W DK 9400265W WO 9501425 A1 WO9501425 A1 WO 9501425A1
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Prior art keywords
protein disulfide
redox agent
disulfide redox
cell
protein
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PCT/DK1994/000265
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English (en)
Inventor
Carsten M. Hjort
Børge DIDERICHSEN
Dorrit Aaslyng
Christian K. Hansen
Jens T÷nne ANDERSEN
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Novo Nordisk A/S
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Application filed by Novo Nordisk A/S filed Critical Novo Nordisk A/S
Priority to AU70683/94A priority Critical patent/AU7068394A/en
Publication of WO1995001425A1 publication Critical patent/WO1995001425A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q5/00—Preparations for care of the hair
    • A61Q5/04—Preparations for permanent waving or straightening the hair
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
    • A23J1/008—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/06—Enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A61K8/66—Enzymes
    • 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
    • C11D3/38636—Preparations containing enzymes, e.g. protease or amylase containing enzymes other than protease, amylase, lipase, cellulase, oxidase or reductase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0036—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0051—Oxidoreductases (1.) acting on a sulfur group of donors (1.8)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/90—Isomerases (5.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y108/00—Oxidoreductases acting on sulfur groups as donors (1.8)
    • C12Y108/01—Oxidoreductases acting on sulfur groups as donors (1.8) with NAD+ or NADP+ as acceptor (1.8.1)
    • C12Y108/01008—Protein-disulfide reductase (1.8.1.8), i.e. thioredoxin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y108/00—Oxidoreductases acting on sulfur groups as donors (1.8)
    • C12Y108/04—Oxidoreductases acting on sulfur groups as donors (1.8) with a disulfide as acceptor (1.8.4)
    • C12Y108/04002—Protein-disulfide reductase (glutathione) (1.8.4.2), i.e. BdbC or BdbD
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y503/00—Intramolecular oxidoreductases (5.3)
    • C12Y503/04—Intramolecular oxidoreductases (5.3) transposing S-S bonds (5.3.4)
    • C12Y503/04001—Protein disulfide-isomerase (5.3.4.1), i.e. disufide bond-forming enzyme
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
    • A61K2800/86—Products or compounds obtained by genetic engineering
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to novel protein disulfide redox agents, DNA sequences expressing these agents, and methods for their production.
  • the invention furthermore relates to compositions comprising
  • compositions can be used for the treatment or degradation of scleroproteins, especially hair, skin and wool, treatment and cleaning of fabrics, as additives to detergents, thickening and gelation of food and fodder, and pharmaceuticals for the alleviation of eye sufferings.
  • protein disulfide redox agents such as protein disulfide reductases, protein disulfide isomerases, protein disulfide oxidases, protein disulfide oxidoreductase, protein disulfide transhydrogenases, sulfhydryl oxidase, and thioredoxins for various purposes has been known for some time.
  • Protein disulfide redox agents catalyses the general reaction:
  • EC 5.3.4.1 (Enzyme Nomenclature, Academic Press, Inc., 1992) refers to an enzyme capable of catalysing the rearrangement of -S-S- bonds in proteins and EC 1.6.4.4 and EC 1.8.4.2 is an example of enzymes catalysing the reaction with NAD(P)H and glutathione as a mediator, respectively.
  • protein disulfide isomerase protein disulfide oxidase, protein sulfhydryl oxidase, protein disulfide reductase, sulfhydryl isomerase, disulfide isomerase, protein disulfide transhydrogenase, protein disulfide oxidoreductase and sulfhydryl oxidase.
  • the protein disulfide redox agents of this invention can be divided into four main groups of enzymes, thioredoxin type (TRX), protein disulfide isomerase type (PDI), disulfide Bond Formation protein type (dsbA) and protein engineered derivatives, chemical modifications and hybrids of TRX and/or PDI and/or dsbA (ENG, sometimes also designated variants or muteins of TRX, PDI or dsbA).
  • TRX thioredoxin type
  • PDI protein disulfide isomerase type
  • dsbA disulfide Bond Formation protein type
  • protein engineered derivatives chemical modifications and hybrids of TRX and/or PDI and/or dsbA (ENG, sometimes also designated variants or muteins of TRX, PDI or dsbA).
  • TRX is a 12-kDa protein having a redox-active disulfide/dithiol and catalysing thiol-disulfide exchange reactions (Edman et al., Nature 317:267-270, 1985; Holmgren, Annu. Rev. Biochem. 54:237-271, 1985; Holmgren, J. Biol. Chem. 264:13963-13966, 1989).
  • PDI consists of two subunits, each consisting of two domains which are homologous to TRX.
  • DsbA is a 21-kDa protein known to be capable of reducing disulfide bonds of insulin and activity common to disulfide oxidoreductases (Bardwell et al., Cell, Vol. 67, 581-589, 1991).
  • TRX, DsbA and the two domains in the subunits of PDI generally comprise a sequence which may be represented as below:
  • TRX and PDI are R 1 and R 2 each different amino acid sequences, X generally is Gly, and Y generally is Pro or His, respectively.
  • TRX from the T 4 -bacteriophage has the sequence: R 1 -Cys-Val-Tyr-Cys-R 2
  • DsbA from E. coll has generally the sequence:
  • a protein disulfide redox agent may therefore be defined as an enzyme exhibiting the above sequence, but where X and Y can be any amino acid residue, and catalysing reaction I above.
  • ENG can be prepared by a variety of methods based on standard recombinant DNA technology:
  • PDI, DsbA, TRX and ENG can be obtained by purification from 1) animal or 2) plant tissues, or from 3) microorganisms, or 4) by expression of recombinant DNA encoding plant, animal, human or microbial PDI, dsbA, TRX or ENG in microorganisms or other suitable hosts, followed by purification of PDI, dsbA, TRX or ENG from extracts or supernatants of said microorganisms.
  • Preparation of TRX according to 1) was described by Luthman and
  • Disulfide linkages in proteins are formed between cysteine residues and have the general function of stabilising the three dimensional structure of the proteins. They can be formed between cysteine residues of the same or different polypeptides.
  • Disulfide linkages are present in many types of proteins such as enzymes, structural proteins, etc.
  • Enzymes are catalytic proteins such as proteases, amylases, etc.
  • structural proteins can be scleroproteins such as keratin, etc, protein material in hair, wool, skin, leather, hides, food, fodder, stains, and human tissue contain disulfide linkages.
  • Treatment of some of these materials with PDI and TRX, and a redox partner has been described previously.
  • TRX for waving, straightening, removing and softening of human and animal hair was described by Pigiet et al. (EP 183506 and WO 8906122).
  • Pigiet (US 4771036) also describes the use of TRX for prevention and reversal of cataracts.
  • Schreiber (DE 2141763 and DE 2141764) describes the use of protein disulfide transhydrogenase for changing the form of human hair.
  • Pigiet (EP 225156) describes the use of TRX for refolding denatured proteins. Use of TRX to prevent metal catalysed oxidative damage in biological reactions is described by Pigiet et al. (EP 237189).
  • Toyoshima et al. (EP 277563 and EP 293793) describe the use of PDI to catalyse renaturation of proteins having reduced disulfide linkages or unnatural oxidised disulfide linkages, in particular in connection with renaturation of recombinantly produced proteins.
  • Brockway (EP 272781), and King and Brockway
  • the present invention relates to novel protein disulfide redox agents, DNA sequences encoding said novel protein disulfide redox agents, DNA constructs comprising said DNA sequences operably linked to sequences capable of directing transcription and translation of said DNA sequence in a suitable host microorganism, and optionally also being operably linked to sequences encoding signals enabling secretion of said protein disulfide redox agents encoded by said DNA sequence to the extracellular medium, microorganisms containing said DNA constructs, and a process for producing protein disulfide redox agents.
  • compositions of matter with improved properties for a number of applications and the use of said compositions in said applications.
  • Said compositions comprise (i) a protein disulfide redox agent optionally in combination with (ii) at least one redox partner, and optionally (iii) one or more other enzymes.
  • Figure 1 displays the plasmid map of pCaHj435 made from the E.Coli expression vector pHD 389 (Lopez - Otin el. al., J. Biol. Chem., in press) comprising the dsbA gen sequence.
  • Figure 2 displays the plasmid map of pPL1759 (Hansen. C., Thesis, The Technical University of Denmark, 1992) .
  • Figure 3 displays the plasmid map of pJA146 made from the pPL1759 plasmid containing the putative mature dsbA encoding region (J.C.A. Bardwell et al., Cell, 67, p. 581-589, 1991).
  • Table 1 shows an alignment of published eukaryotic PDI amino acid sequences: Bovine (Bos taurus) (Yamauchi et al., Biochem. Biophys. Res. Commun. 146:1485-1492, 1987), chicken (Gallus gallus) (Parkkonen et al., Biochem. J. 256:1005-1011, 1988), human (Homo sapiens) (Rapilajaniemi et al. EMBO J. 6:643-649, 1987), mouse (Mus musculus) (Gong, et al., Nucleic Acids Res. 16:1203, 1988), rabbit (Oryctolagus cuniculus) (Fliegel et al., J. Biol.
  • Table 2 shows an alignment of PDI amino acid sequences: Alfalfa (Medicago sativa) (Shorrosh and Dixon, Plant. Mol. Bio. 19:319- 321, 1992), A. oryzae, yeast (Saccharomyces cerevisiae) (Tachi-kawa et al., J. Biochem. 110:306-313), bovine (Bos taurus) (Yamauchi et al., Biochem. Biophys. Res. Commun.
  • novel protein disulfide redox agents of the invention comprises agents of the type ENG indicated above, wherein one or more of the sequences
  • R 1 -Cys-X-Y-Cys-R 2 (A) has been changed to the following definition:
  • R 1 and R 2 each independently are different amino acid sequences identical to or different from the corresponding sequences in their parent molecule
  • X and Y each independently are any of the 20 other naturally occurring amino acids different from the ones in their parent molecule with the exception of TRX from E. coli , wherein His has been substituted for Pro as Y (E. coll P34H), and DsbA from E. coli .
  • R 1 and R 2 are preferably of a length between 5 and 500 amino acid residues.
  • X is chosen among Ala, Val, Leu, Ile, Pro, Phe, Trp, Met, Gly, Ser, Thr, Cys, Tyr, Asn, Gln, Asp, Glu, Lys, Arg and His
  • Y is chosen among Ala, Val, Leu, Ile, Pro, Phe, Trp, Met, Gly, Ser, Thr, Cys, Tyr, Asn, Gln, Asp, Glu, Lys, Arg and His.
  • X is chosen among Glu, Ala, Ser, Asp, Pro, Val and Y is chosen among Arg, Lys, Asn, Gln or Tyr.
  • X is Gly, Val or Ala
  • Y is any of the 20 other naturally occurring amino acids different from the ones in their parent molecule, preferably Arg, Lys, Asn, Gln, Tyr or His.
  • Y is either His, Pro or Tyr
  • X is any of the 20 other naturally occurring amino acids different from the ones in their parent molecule, preferably Glu, Ala, Ser, or Asp.
  • the parent molecule is divided or truncated so as to provide for a smaller number of sequences (A) in each peptide, e.g. one PDI subunit with 2 CA) sequences or a split PDI, dsbA or TRX unit with only one (A) sequence; or wherein R 1 and R 2 different from those of the parent molecule.
  • R and R 2 may be made both shorter or longer.
  • the novel protein disulfide redox agents of the invention can be obtained through the methods indicated below.
  • the invention also relates to DNA sequences coding for the variants of the invention.
  • the DNA sequence of the DNA construct of the invention may be isolated by well-known methods.
  • the DNA sequence may, for instance, be isolated by establishing a cDNA or genomic library from an organism expected to harbour the sequence, and screening for positive clones by conventional procedures. Examples of such procedures are hybridization to oligonucleotide probes synthesized on the basis of any of the full amino acid sequences shown in Tables 1 and 2, or a subsequence thereof in accordance with standard techniques (cf.
  • a preferred method of isolating a DNA construct of the invention from a cDNA or genomic library is by use of polymerase chain reaction (PCR) using degenerate oligonucleotide probes prepared on the basis of the amino acid sequence of the parent protein disulfide redox agent of the invention.
  • PCR polymerase chain reaction
  • the PCR may be carried out using the techniques described in US Patent No. 4,683,202 or by R.K. Saiki et al. (1988).
  • the DNA sequence of the DNA construct of the invention may be prepared synthetically by established standard methods, e.g. the phosphoamidite method described by Beaucage and Caruthers (1981), or the method described by Matthes et al.
  • oligonucleotides are synthesized, e.g. in an automatic DNA synthesizer, purified, annealed, ligated and cloned in appropriate vectors.
  • the DNA construct may be of mixed genomic and synthetic, mixed synthetic and cDNA or mixed genomic and cDNA origin prepared by ligating fragments of synthetic, genomic or cDNA origin (as appropriate), the fragments corresponding to various parts of the entire recombinant DNA molecule, in accordance with standard techniques. Generation of random mutations in the protein disulfide redox agent gene
  • protein disulfide redox agent gene has been cloned into a suitable vector, such as a plasmid, several methods can be used to introduce random mutations into the gene.
  • One method would be to incorporate the cloned protein disulfide redox agent gene, as part of a retrievable vector, into a mutator strain of Eschericia coli .
  • Another method would involve generating a single stranded form of the protein disulfide redox agent gene, and then annealing the fragment of DNA containing the protein disulfide redox agent gene with another DNA fragment such that a portion of the protein disulfide redox agent gene remained single stranded.
  • This discrete, single stranded region could then be exposed to any of a number of mutagenizing agents, including, but not limited to, sodium bisulfite, hydroxylamine, nitrous acid, formic acid, or hydralazine.
  • a specific example of this method for generating random mutations is described by Shortle and Nathans (1978, Proc. Natl. Acad. Sci. U.S.A., 75: 2170-2174).
  • the plasmid bearing the protein disulfide redox agent gene would be nicked by a restriction enzyme that cleaves within the gene. This nick would be widened into a gap using the exonuclease action of DNA polymerase I. The resulting single-stranded gap could then be mutagenized using any one of the above mentioned mutagenizing agents. Generation of site directed mutations in the protein disulfide redox agent gene
  • these mutations can be introduced using synthetic oligonucleotides. These oligonucleotides contain nucleotide sequences flanking the desired mutation sites; mutant nucleotides are inserted during oligonucleotide synthesis.
  • a single stranded gap of DNA, bridging the protein disulfide redox agent gene is created in a vector bearing the protein disulfide redox agent gene. Then the synthetic nucleotide, bearing the desired mutation, is annealed to a homologous portion of the single-stranded DNA.
  • the remaining gap is then filled in by DNA polymerase I (Klenow fragment) and the construct is ligated using T4 ligase.
  • DNA polymerase I Klenow fragment
  • T4 ligase A specific example of this method is described in Morinaga et al., (1984, Biotechnology 2:646-639). According to Morinaga et al., a fragment within the gene is removed using restriction endonuclease. The vector/gene, now containing a gap, is then denatured and hybridized to a vector/gene which, instead of containing a gap, has been cleaved with another restriction endonuclease at a site outside the area involved in the gap.
  • a single-stranded region of the gene is then available for hybridization with mutated oligonucleotides, the remaining gap is filled in by the Klenow fragment of DNA polymerase I, the insertions are ligated with T4 DNA ligase, and, after one cycle of replication, a double-stranded plasmid bearing the desired mutation is produced.
  • the Morinaga method obviates the additional manipulation of constructing new restriction sites, and therefore facilitates the generation of mutations at multiple sites.
  • a mutated protein disulfide redox agent gene produced by methods described above, or any alternative methods known in the art can be expressed, in enzyme form, using an expression vector.
  • An expression vector generally falls under the definition of a cloning vector, since an expression vector usually includes the components of a typical cloning vector, namely, an element that permits autonomous replication of the vector in a microorganism independent of the genome of the microorganism, and one or more phenotypic markers for selection purposes.
  • An expression vector includes control sequences encoding a promoter, operator, ribosome binding site, translation initiation signal, and, optionally, a repressor gene or various activator genes.
  • nucleotides encoding a "signal sequence" may be inserted prior to the coding sequence of the gene.
  • a target gene to be treated according to the invention is operably linked to the control sequences in the proper reading frame.
  • Promoter sequences that can be incorporated into plasmid vectors, and which can support the transcription of the mutant protein disulfide redox agent gene include but are not limited to the prokaryotic ⁇ -lactamase promoter (Villa-Kamaroff, et al., 1978, Proc. Natl. Acad. Sci. U.S.A.
  • the expression vector carrying the DNA construct of the invention may be any vector which may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced.
  • the vector may be an autonomously replicating vector, i.e. a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid, a bacteriophage or an extrachromosomal element, minichromosome or an artificial chromosome.
  • the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome (s) into which it has been integrated.
  • the DNA sequence should be operably connected to a suitable promoter sequence.
  • the promoter may be any DNA sequence which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell.
  • suitable promoters for directing the transcription of the DNA construct of the invention, especially in a bacterial host are the promoter of the lac operon of E.
  • the Streptomyces coelicolor agarase gene dagA promoters the promoters of the Bacillus licheniformis ⁇ -amylase gene (amyL), the promoters of the Bacillus stearothermophilus maltogenic amylase gene (amyM), the promoters of the Bacillus Amyloliquufaciens ⁇ -amylase (amyQ) , the promoters of the Bacillus subtilis xylA and xylB genes etc.
  • useful promoters are those derived from the gene encoding A .
  • oryzae TAKA amylase Rhizomucor miehei aspartic proteinase, A . niger neutral ⁇ -amylase, A. niger acid stable ⁇ -amylase, A. niger glucoamylase, Rhizomucor miehei lipase, A . oryzae alkaline protease, A . oryzae triose phosphate isomerase or A. nidulans acetamidase.
  • the expression vector of the invention may also comprise a suitable transcription terminator and, in eukaryotes, polyadenylation sequences operably connected to the DNA sequence encoding the protein disulfide redox agent of the invention. Termination and polyadenylation sequences may suitably be derived from the same sources as the promoter.
  • the vector may further comprise a DNA sequence enabling the vector to replicate in the host cell in question.
  • a DNA sequence enabling the vector to replicate in the host cell in question. Examples of such sequences are the origins of replication of plasmids pUC19, pACYC177, pUBHO, pE194, pAMBl, pHD 389 and pIJ702.
  • the vector may also comprise a selectable marker, e.g. a gene the product of which complements a defect in the host cell, such as the dal genes from B. subtilis or B . licheniformis, or one which confers antibiotic resistance such as ampicillin, kanamycin, chloramphenicol or tetracyclin resistance.
  • a selectable marker e.g. a gene the product of which complements a defect in the host cell, such as the dal genes from B. subtilis or B . licheniformis, or one which confers antibiotic resistance such as ampicillin, kanamycin, chloramphenicol or tetracyclin resistance.
  • Aspergillus selection markers include amdS, argB, niaD and sC, a marker giving rise to hygromycin resistance.
  • the selection may be accomplished by co-transformation, e.g. as described in WO 91/17243.
  • the protein disulfide redox agents of the invention comprising a variant of any of the amino acid sequences shown in tables 1 or 2 may furthermore comprise a preregion permitting secretion of the expressed protein disulfide isomerase into the culture medium. If desirable, this preregion may be native to the protein disulfide isomerase of the invention or substituted with a different preregion or signal sequence, conveniently accomplished by substitution of the DNA sequences encoding the respective preregions.
  • the procedures used to ligate the DNA construct of the invention, the promoter, terminator and other elements, respectively, and to insert them into suitable vectors containing the information necessary for replication, are well known to persons skilled in the art (cf., for instance, Sambrook et al. (1989)).
  • the cell of the invention either comprising a DNA construct or an expression vector of the invention as defined above is advantageously used as a host cell in the recombinant production of a polypeptide of the invention.
  • the cell may be transformed with the DNA construct of the invention, conveniently by integrating the DNA construct in the host chromosome. This integration is generally considered to be an advantage as the DNA sequence is more likely to be stably maintained in the cell.
  • Integration of the DNA constructs into the host chromosome may be performed according to conventional methods, e.g. by homologous or heterologous recombination.
  • the cell may be transformed with an expression vector as described above in connection with the different types of host cells.
  • the cell of the invention may be a cell of a higher organism such as a mammal, an avian, an insect, or a plant cell, but is preferably a microbial cell, e.g. a bacterial or a fungal (including yeast) cell.
  • a microbial cell e.g. a bacterial or a fungal (including yeast) cell.
  • suitable bacteria are gram positive bacteria such as Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillus amyloli ⁇ juefaciens, Bacillus coagulans, Bacillus circulans, Bacillus lautus, Bacillus megaterium, Ba- cillus thuringiensis, or Streptomyces lividan ⁇ or Streptomyces murinus, or gram negative bacteria such as E. coli .
  • the transformation of the bacteria may for instance be effected by protoplast transformation or by using competent cells in a manner known per se .
  • the yeast organism may favourably be selected from a species of Saccharomyces or Schizosaccharomyces, e.g. Saccharomyces cerevisiae .
  • the filamentous fungus may advantageously belong to a species of Aspergillus, e.g. Aspergillus oryzae or Aspergil lus niger.
  • a strain of a Fusarium species e.g. F. oxysporum, can be used as a host cell.
  • Fungal cells may be transformed by a process involving protoplast formation and transformation of the protoplasts followed by regeneration of the cell wall in a manner known per se . A suitable procedure for transformation of Aspergillus host cells is described in EP 238 023.
  • a suitable method of transforming Fusarium species is described by Malardier et al., 1989.
  • expression of the DNA construct comprising the recombinant DNA sequence or expression vector carrying the DNA construct may take place as heterologous expression in a host cell different from the cell from where the recombinant DNA was derived.
  • prokaryote recombinant DNA may take place heterologously in cell compartments.
  • the recombinant DNA derived from a cell e.g. of the genus Escherichia can be expressed in an other cell e.g. of the genus Bacillus or Streptomyces .
  • the recombinant DNA derived from a cell e.g. of the genus Aspergillus can be expressed in a cell e.g. of the genus Bacillus or Streptomyces .
  • the present invention relates to a method of producing a recombinant protein disulfide redox agent of the invention, which method comprises cultivating a host cell as described above under conditions conducive to the production of the protein disulfide redox agent and recovering the protein disulfide redox agent from the cells and/or culture medium.
  • the medium used to cultivate the cells may be any conventional medium suitable for growing the host cell in question and obtaining expression of the protein disulfide redox agent of the invention. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g. in catalogues of the American Type Culture Collection).
  • the resulting protein disulfide redox agent may be recovered from the medium by conventional procedures including separating the cells from the medium by centrifugation or filtration, if necessary after disruption of the cells, precipitating the proteinaceous components of the supernatant or filtrate by means of a salt, e.g. ammonium sulphate, followed by purification by a variety of chromatographic procedures, e.g. ion exchange chromatography, affinity chromatography, or the like. It is of course also possible to produce the protein disulfide redox agent of the invention by culturing the filamentous fungal natural host or parent organism of interest and recovering the protein disulfide isomerase from the culture broth in traditional ways.
  • a salt e.g. ammonium sulphate
  • the present invention also relates to compositions comprising the protein disulfide redox agents of the invention, including also the variant of TRX from E. coli , wherein His has been substituted for Pro as Y (E. coli P34H).
  • compositions may suitably contain 0.01-200 mg of enzyme protein per gram, preferably 0.01-20 mg of enzyme protein per gram, especially 0.01-2 mg of enzyme protein per gram, or alternatively 0.02-0.2 mg of enzyme protein per gram, or 0.01-0.2 mg of enzyme protein per gram.
  • the composistion contain 0.01-0.5 mg of enzyme protein per gram or conversly 0.2-0.5 mg of enzyme protein per gram.
  • compositions of the invention usually also comprises (ii) a suitable redox partner.
  • the redox partner (ii) is generally an organic or inorganic reductant, and would often be selected from the organic reductants, such as from the group comprising glutathione, L-cysteine, dithiothreitol, 2-mercaptoethanol, thioglycolic acid, L-cysteine ethylester, ⁇ -mercaptoethylamine, mercaptosuccinic acid, ⁇ -mercaptopropionic acid, dimercapto adipic acid, thiomalic acid, thioglycolamides, glycol thioglycolate, glycerol thioglycolate, thiolactic acid and salts thereof.
  • organic reductants such as from the group comprising glutathione, L-cysteine, dithiothreitol, 2-mercaptoethanol, thioglycolic acid, L-cysteine ethylester, ⁇ -mercaptoethylamine,
  • the invention is not meant to comprise compositions comprising only naturally occurring TRX or PDI in combination with a redox partner. All types of ENG are naturally encompassed by the present invention also under this aspect.
  • compositions of the invention may optionally comprise (iii) another enzyme, where said other enzyme preferably is selected among proteases, lipases, amylases, transglutaminases, or another protein disulfide redox agent
  • compositions comprising all types of protein disulfide redox agents including naturally occurring TRX or PDI either without or in combination with a redox partner. All types of ENG are nat urally encompassed by the present invention also under this aspect.
  • compositions of the invention may contain other ingredients known in the art as e.g. excipients, stabilizers, fillers, detergents, etc.
  • compositions of the invention may be formulated in any convenient form, e.g. as a powder, paste, liquid or in granular form.
  • the enzyme may be stabilized in a liquid by inclusion of enzyme stabilizers.
  • the pH of a solution of the composition of the invention will be 5-10 and in some instances 7.0-8.5.
  • Other enzymes such as proteases, cellulases, oxidases, peroxidases, amylases or lipases may be included in the compositions of the invention, either separately or in a combined additive.
  • compositions of the invention can be used for the treatment or degradation of scleroproteins, especially hair, skin and wool, treatment and cleaning of fabrics, as additives to detergents, thickening and gelation of food and fodder, strengthening of gluten in bakery or pastry products, and as pharmaceuticals for the alleviation of eye sufferings.
  • the present invention is further illustrated in the following examples which should not, in any manner, be considered to limit the scope of the present invention.
  • E. coli WA803 Maniatis et al., 1982, Molecular cloning, a laboratory manual, Cold Spring Habor Laboratory, New York
  • Subtilis DN1885 P. L. Joergensen et al., Gene, 96, p. 37-41, 1990
  • JA146 B . subtilis DN1885 harbouring the pJA146 plasmid CaHj435 : E. coli harbouring the pCaHj435 plasmid Plasmids :
  • pCaHj435 Figure 1, plasmid comprising the dsbA gene sequence in E. Coli expression vector pHD389.
  • pJA146 Figure 3 plasmid comprising the putative dsbA encoding region (J.C.A. Bardwell et al. Cell, 67, p. 581-589, 1991) in B . subtilis expression vector
  • pPL1759 B . subtilis expression vector (Hansen C., 1992, Thesis, The Technical University of Denmark), figure 2.
  • pHD389 E. coli expression vector, (Lopez - Otin et al., J. Biol. Chem., in press)
  • LB agar Lia-Bertani medium/agar, CR. Harwood and S.M.
  • N-terminal amino acid sequence analysis of recombinant dsbA was carried out following electroblotting using an Applied Biosystems 473A protein sequencer operated according to the manufacturers instructions.
  • the E. coli dsbA gene sequence was collected from the GenBank database (accession number M77746). Based on this sequence a PCR primer containing the restriction enzyme Cla I recognition sequence and 23 bases of the dsbA 5' coding sequence (primer 5513) and a PCR primer containing the restriction enzyme Sail recognition sequence and 23 bases complementary to the dsbA 3' coding sequence (primer 5512) were constructed.
  • Total DNA was extracted from E. coli strain WA803 using standard procedures .
  • This DNA was used without further modification as template in a PCR reaction (20 cycles) using the primers 5513 and 5512 and the Perkin Elmer- cetus AmplitaqTM Taq polymerase following the manufacturer's instructions.
  • a PCR fragment corresponding to the size of the dsbA gene was recovered from an agarose gel and digested with the restriction enzymes Clal and Sail.
  • the E. coli expression vector pHD 389 was digested with the same enzymes, and the large vector fragment was ligated to the digested PCR fragment.
  • the ligation mixture was used to transform E. coli strain WA803. After 24 hours of growth at 30°C using ampicillin selection a transformant was selected and subsequent DNA sequence analysis using the DNA sequencing kit SequenaseTM showed that a sequence identical to the published dsbA gene sequence was integrated between the lambda PR promoter and the fd terminator.
  • This plasmid was termed pCaHj 435, and the E. coli strain harbouring the plasmid was termed CaHj 435.
  • a plasmid map of pCaHj 435 is shown in figure 1. Expression of dsbA in E. coli phage lambda
  • the dsbA gene is under control of the promoter PR from the E. coli phage lambda. PR is repressed by CI repressor also harboured by the plasmid pCaHj 435. However the CI repressor allele used in this plasmid is temperature sensitive being active at 30°C but inactive at 42°C. Thus the dsbA gene is repressed at 30°C but expressed at 42°C.
  • the strain CaHj 435 was grown in shake flasks containing the medium Terrific Broth at 30°C and 200 rpm until OD600 reached 0.2. Then the shake flasks were transferred to 42°C (200 rpm) for 18 hours.
  • the cells were isolated by centrifugation (2500 ⁇ g, 15 min.) and resuspended in 100 ml 20% (W/V) sucrose buffered with 10 mM Tris/HCl pH 7.0. EDTA were added to a final concentration of 15 mM.
  • the cell suspension was incubated on ice for 15 min. and then the cells were collected by centrifugation (2500 ⁇ g, 15 min.).
  • the cells were resuspended in 70 ml of water by vigorous shaking and subsequently incubated on ice (10 min).
  • the suspension was centrifuged (2500 ⁇ g, 15 min.) and the supernatant containing the soluble periplasmic fraction was isolated. Tris/HCl pH 7.0 was added to a final concentration of 5 mM.
  • the dsbA gene product was then purified by DEAE anion exchange chromatography.
  • a column containing 20 ml of DEAE Sephadex A-50 purchased from Pharmacia Fine Chemicals AB was equilibrated with 10 mM Tris/HCl pH 7.0.
  • the osmotic shock preparation was applied to the column, and then the column was washed with 200 ml 10 mM Tris/HCl pH 7.0.
  • the dsbA gene product was eluted with 50 ml 50 mM NaCl, 10 mM Tris/HCl pH 7.0.
  • SDS polyacrylamide gel electrophoresis showed that more than 90% of the protein isolated corresponded to the size of the dsbA gene.
  • the E. coli dsbA gene sequence was collected from the GenBank database (acession number M 77746 ) . Based on the dsbA sequence from GenBank and pCaHj435 (the dsbA expression plasmid in E. coli (WA803)) a PCR primer containing the restriction enzyme Nsil recognition sequence and 27 bases of the dsbA 5'sequence encoding the putative N-terminal (J.C.A. Bardwell et al. Cell, 67, p.
  • Super TaqTM DNA polymerase Super TaqTM DNA polymerase
  • DNA analysis of the plasmids from those cells using a DNA sequencing Kit showed the expected sequence of the promotor- and signal peptide encoding regions of amyL (P.L. Joergensen et al., Gene, 96, p. 37-41, 1990) fused to the above mentioned putative mature dsbA encoding region.
  • This plasmid was termed pJA146 and a B . subtilis DN1885 strain haboring this plasmid was termed JA146.
  • a plasmid map of pJAl46 is shown in fig. 3.
  • Strain JA146 was grown for 18 hours in Terrific Yeast medium at
  • N-terminal amino acid sequence was analysed as described above.
  • the N-terminal amino acid of DsbA determined was : Ala-Ala-Gln-Tyr-Glu-Asp-Gly-Lys-Gln- Example 3
  • a tress of washed human Scandinavian hair (1 gram) was wetted with water and tightly wound on a curling roller. 1 ml of a solution with the following composition and a temperature of 30°C was applied to the tress:
  • the tress was put in a plastic bag and incubated for 60 minutes at 30°C. Then the roller was removed and the hair was rinsed with water, dried with a cotton towel, combed and air dried. Other tresses of hair was treated like above but without addition of the P34H Thioredoxin variant.
  • Tresses of washed brown and fair human European hair (1 gram) was wetted with water and tightly wound on curling rollers. 1 ml of waving solution with the following composition and temperature of 30°C was applied to the tresses.
  • Tresses were put in plastic bags and stored for 60 minutes at 30°C.
  • the hair was rinsed with water, dried with cotton towel, the rollers was removed, the hair was air dried and combed.
  • control tresses and enzyme treated hair tresses were washed in either water or a mild shampoo, rinsed and air dried.
  • GFPTIKLFAA GAKDSPVEYE

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Abstract

Nouveaux agents oxydo-réducteurs de bisulphure protéique, séquences d'ADN exprimant ces agents et procédés d'obtention. En outre, l'invention se rapporte à des compositions renfermant: (i) un agent oxydo-réducteur de bisulphure protéique en combinaison avec (ii) au moins un partenaire oxydo-réducteur et, facultativement (iii) au moins un ou plusieurs autres enzymes. Ces compositions peuvent servir au traitement ou à la dégradation des scléroprotéines, notamment des cheveux, de la peau et de la laine, au traitement et au nettoyage des tissus, comme additifs dans des détergents, comme agents épaississeurs ou gélifiants de produits alimentaires et de fourrages et comme substances pharmaceutiques destinées à soulager les douleurs oculaires.
PCT/DK1994/000265 1993-06-28 1994-06-28 Compositions renfermant des agents oxydo-reducteurs de bisulphure proteique WO1995001425A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996019590A1 (fr) * 1994-12-21 1996-06-27 Novo Nordisk A/S Procede d'epilage de cuirs et de peaux au moyen d'enzymes
WO2000070064A1 (fr) * 1999-05-17 2000-11-23 Novozymes A/S Polypeptides reducteurs des bisulfures de proteines
US7009087B1 (en) 2000-12-01 2006-03-07 Pioneer Hi-Bred International, Inc. Compositions and methods for altering the disulfide status of proteins
US7238513B2 (en) 2003-02-28 2007-07-03 University Of Utah Research Foundation Nucleic acid sequences encoding Conus protein disulfide isomerase

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE10059748A1 (de) * 2000-12-01 2002-06-20 Henkel Kgaa Enzymatische Fixierung von derivatisierten Kohlenhydraten an fasrigen Materialien
WO2016175577A1 (fr) * 2015-04-30 2016-11-03 조선대학교 산학협력단 Composition d'agent oxydant de permanente contenant une enzyme
CN114763552B (zh) * 2021-01-12 2024-04-30 山东大学 一种微生物转谷氨酰胺酶的重组生产方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE2141763A1 (de) * 1971-08-20 1973-03-01 Henkel & Cie Gmbh Mittel zur formveraenderung von skleroproteinen
DE2141764A1 (de) * 1971-08-20 1973-03-01 Henkel & Cie Gmbh Mittel zur formveraenderung von skleroproteinen
EP0272781A1 (fr) * 1986-11-04 1988-06-29 The University Of Reading Traitement de la chevelure

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1988005816A1 (fr) * 1987-01-28 1988-08-11 Takeda Chemical Industries, Ltd. Polypeptide

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE2141763A1 (de) * 1971-08-20 1973-03-01 Henkel & Cie Gmbh Mittel zur formveraenderung von skleroproteinen
DE2141764A1 (de) * 1971-08-20 1973-03-01 Henkel & Cie Gmbh Mittel zur formveraenderung von skleroproteinen
EP0272781A1 (fr) * 1986-11-04 1988-06-29 The University Of Reading Traitement de la chevelure

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
DIALOG INFORMATION SERVICES, File 155, Medline, Dialog Accession No. 07246967, JOELSON T. et al., "Modifications of the Active Center of T4 Thioredoxin by Site-Directed Mutagenesis"; & J. BIOL. CHEM., (UNITES STATES), 25 Feb. 1990, 265(6), p3183-8. *
DIALOG INFORMATION SERVICES, File 155, Medline, Dialog Accession No. 07635193, KRAUSE G. et al., "Substitution of the Conserved Tryptophan 31 in Escherichia Coli Thioredoxin by Site-Directed Mutagenesis and Structure-Function Analysis"; & J. BIOL. CHEM., (UNITED STATES), 5 Mar. 1991, 266(7), p4056-66. *
DIALOG INFORMATION SERVICES, File 155, Medline, Dialog Accession No. 08112497, LUNDSTROEM J. et al., "A Pro to His Mutation Inactive Site of Thioredoxin Increases its Disulfide Isomerase Activity 10 Fold. New Refolding Systems for Reduced or Randomly Oxidized Ribonuclease"; & J. BIOL. CHEM., (UNITED STATES), 5 May 1992, *
DIALOG INFORMATION SERVICES, File 155, Medline, Dialog Accession No. 08554420, NIKKOLA M. et al., "Crystal Structure Analysis of a Mutant Escherichia Coli Thioredoxin in Which Lysine 36 is Replaced by Glutamic Acid"; & BIOCHEMISTRY (UNITED STATES), 18 May 1993, 32 (19), p5093-8. *
J. BASIC MICROBIOL., Volume 33, No. 3, 1993, PETER MINARIK, "Influence of the Different Amino Acid Substitutions in Escherichia Coli Thioredoxin on the Growth of Bacteriophages T7 and f1", pages 213-215. *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996019590A1 (fr) * 1994-12-21 1996-06-27 Novo Nordisk A/S Procede d'epilage de cuirs et de peaux au moyen d'enzymes
AU693981B2 (en) * 1994-12-21 1998-07-09 Novo Nordisk A/S Method for dehairing of hides or skins by means of enzymes
US5834299A (en) * 1994-12-21 1998-11-10 Novo Nordisk A/S Method for dehairing of hides or skins by means of enzymes
WO2000070064A1 (fr) * 1999-05-17 2000-11-23 Novozymes A/S Polypeptides reducteurs des bisulfures de proteines
US7009087B1 (en) 2000-12-01 2006-03-07 Pioneer Hi-Bred International, Inc. Compositions and methods for altering the disulfide status of proteins
US7465852B2 (en) 2000-12-01 2008-12-16 Pioneer Hi-Bred International, Inc. Compositions and methods for altering the disulfide status of proteins
US7238513B2 (en) 2003-02-28 2007-07-03 University Of Utah Research Foundation Nucleic acid sequences encoding Conus protein disulfide isomerase

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