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WO2003038084A1 - Ascopyrone p synthase - Google Patents

Ascopyrone p synthase Download PDF

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Publication number
WO2003038084A1
WO2003038084A1 PCT/GB2002/004885 GB0204885W WO03038084A1 WO 2003038084 A1 WO2003038084 A1 WO 2003038084A1 GB 0204885 W GB0204885 W GB 0204885W WO 03038084 A1 WO03038084 A1 WO 03038084A1
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WO
WIPO (PCT)
Prior art keywords
ascopyrone
synthase
sequence
sequences
present
Prior art date
Application number
PCT/GB2002/004885
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English (en)
Inventor
Andrew John Morgan
Charlotte Refdahl
Shukun Yu
Original Assignee
Danisco A/S
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from GBGB0126163.5A external-priority patent/GB0126163D0/en
Application filed by Danisco A/S filed Critical Danisco A/S
Publication of WO2003038084A1 publication Critical patent/WO2003038084A1/fr

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    • 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
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/02—Oxygen as only ring hetero atoms
    • C12P17/06—Oxygen as only ring hetero atoms containing a six-membered hetero ring, e.g. fluorescein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl group
    • C12P7/26—Ketones

Definitions

  • the present invention relates to the purification and characterisation of ascopyrone P synthase.
  • Ascopyrone P is a good antioxidant, antibrowning agent and antiftricrobial [WO 00/56838 filed 16/3/00, " claiming priority from GB9906457.8, filed 19/3/99; WO 02/26060 filed 27/9/01, claiming priority from GB0023686.9 and GB0023687.7, both filed 27/9/00].
  • APP was first prepared from amylopectin, amylose and cellulose by pyrolysis, but the yield of APP was less than 3.0 % [Shaf ⁇ zadeh, F., Furneaux R.H., Stevenson, T.T., and Cochran, T.G., Carbohydr. Res.
  • APP was later isolated from the fungi of the order Pezizales, such as Anihracobia melaloma, Plicaria anthracina, P. leiocarpa, and Peziza petersi [M.-A. Baute, G. Deffieux, J. Nercauteren, R. Baute, and A. Badoc, Phytochemistry, 33 (1993): 41-45].
  • AFDH anhydrofructose dehydratase
  • APS ascopyrone P synthase
  • Scheme 1 illustrates the proposed formation of ascopyrone P (APP) from starch-typed substrates (starch, dextrins, maltosaccharides, and glycogen etc.).
  • the reactions catalyzed are: 1, ⁇ -l,4-glucan lyase (EC 4.2.2.13); 2, 1,5-anhydro-D-fructose dehydratase, and 3, APP synthase (enolone or ketoenol isomerase, enolone or ketoenol tautomerase).
  • the invention relates to the purification and characterisation of ascopyrone P synthase.
  • expression As used with reference to the present invention, the terms "expression”, “expresses”, “expressed” and “expressable” are synonymous with the respective terms “transcription”, “transcribes”, “transcribed” and “transcribable”.
  • nucleotide sequence of the present invention includes: a construct comprising the sequences of the present invention; a vector comprising the sequences of the present invention; a plasmid comprising the sequences of present invention; a transformed cell comprising the sequences of the present invention; a transformed tissue comprising the sequences of the present invention; a transformed organ comprising the sequences of the present invention; a transformed host comprising the sequences of the present invention; a transformed organism comprising the sequences of the present invention.
  • the present invention also encompasses methods of expressing the nucleotide sequence using the same, such as expression in a host plant cell; including methods for transferring same.
  • the invention relates to ascopyrone P synthase in isolated or purified form or comprising at least one amino acid sequence selected from: (i) AINLPFSNWAX(or C)TI; and (ii) EYGRTFFTRYDYENVD.
  • the invention relates to ascopyrone P synthase in isolated or purified form which has an optimim temperature range of 25 to 50 °C.
  • the nucleotide sequence is obtainable from Anthracobia melaioma.
  • the ascopyrone P synthase of the invention has an optimum temperature of about 48 °C.
  • the ascopyrone P synthase of the invention has an optimal pH range of from about 4.5 to 7.5.
  • the optimal pH range is from about 5.0 to 6.0.
  • the ascopyrone P synthase has an optimal pH of about 5.5.
  • the ascopyrone P synthase of the invention is stable in 50 mM sodium phosphate buffer (pH 7.0) containing 0.1 M NaCl for at least one week at 4 °C.
  • the ascopyrone P synthase of the invention is stable in 50 mM sodium phosphate buffer (pH 7.0) containing 0.1 M NaCl for at least one month at 4 °C.
  • (iii) is stable in 50 mM sodium phosphate buffer (pH 7.0) containing 0.1 M NaCl for at least one week at 4 °C.
  • the ascopyrone P synthase of the invention has the following characteristics:
  • (iii) is stable in 50 mM sodium phosphate buffer (pH 7.0) containing 0.1 M NaCl for at least one week at 4 °C.
  • (iii) is stable in 50 mM sodium phosphate buffer (pH 7.0) containing 0.1 M NaCl for at least one week at 4 °C.
  • (iii) is stable in 50 mM sodium phosphate buffer (pH 7.0) containing 0.1 M NaCl for at least one week at 4 °C.
  • the ascopyrone P synthase of the invention has the following characteristics: (i) an optimum temperature of about 48 °C; (ii) an optimal pH of about 5.5 ; and (iii) is stable in 50 mM sodium phosphate buffer (pH 7.0) containing 0.1 M NaCl for at least one week at 4 °C.
  • the ascopyrone P synthase of the invention is in the form of a homodimer.
  • the present invention also encompasses different isoforms of the ascopyrone P synthase described herein.
  • the term "isoform” refers to a protein having the same function (namely ascopyrone P synthase activity), which has a similar or identical amino acid sequence, but which is the product of a different gene.
  • APS1 and APS2 hydrophobic interaction chromatography
  • APS1 into 3 isoforms using ion-exchange chromatography step. Further details of the isoforms may be found in the accompanying examples.
  • the ascopyrone P synthase comprises an amino acid sequence selected from AINLPFSNWAX(or C)TI and EYGRTFFTRYDYENND.
  • a further aspect provides a process for preparing ascopyrone P using the ascopyrone P synthase of the invention.
  • the process further comprises the use of 1,5-anhydro-D- fructose dehydratase in the preparation of ascopyrone P.
  • the process comprises contacting 1,5-anhydro-D-fructose dehydratase and the ascopyrone P synthase of the invention with 1,5-anhydro-D-fructose.
  • the process further comprises the use of ⁇ -l,4-glucan lyase.
  • the process comprises contacting ⁇ -l,4-glucan lyase, 1,5-anhydro-D-fructose dehydratase and the ascopyrone P synthase of the invention with a starch-type substrate.
  • starch-type substrate includes, for example, glycogen, or an intermediate compound resulting from the hydrolysis of starch by amylase enzymes, such as a maltodextrin.
  • amylase enzymes such as a maltodextrin.
  • starch-type substrates include starch, amylopectin, amylose and dextrin.
  • the starch-type substrate is selected from glycogen or a maltodextrin.
  • the process comprises the steps of:
  • step (ii) contacting the product from step (i) with 1,5-anhydro-D-fructose dehydratase and the ascopyrone P synthase of the invention.
  • Another aspect of the invention relates to a process for converting a compound of formula I into a compound of formula II
  • Yet another aspect of the invention relates to a process for converting a compound of formula II into a compound of formula I
  • the APP synthase used in converting said compound of formula I into said compound of formula II (or vice versa) is as defined hereinbefore.
  • Ri and R are linked together to form a cyclic structure.
  • the present invention relates to the purification and characterisation of ascopyrone P synthase. To date, this enzyme has neither been isolated nor purified.
  • the enzyme and sequence of the present invention may be used in the production of APP.
  • APP is itself useful as, inter alia, an anti-microbial material.
  • the following assay may be used to characterise and identify actual and putative amino acid sequences according to the present invention.
  • the sequence is in an isolated form.
  • isolated means that the sequence is not in its natural environment (i.e. as found in nature).
  • isolated means that the sequence is at least substantially free from at least one other component with which the sequence is naturally associated in nature and as found in nature.
  • the sequence may be separated from at least one other component with which it is naturally associated.
  • the sequence is in a purified form.
  • purified also means that the sequence is not in its naural environment (i.e. as found in nature).
  • purified means that the sequence is at least substantially separated from at least one other compnent with which the sequence is naturally associated in nature and as found in nature.
  • nucleotide sequence refers to an oligonucleotide sequence or polynucleotide sequence, and variant, homologues, fragments and derivatives thereof (such as portions thereof).
  • the nucleotide sequence may be of genomic or synthetic or recombinant origin, which may be double- stranded or single-stranded whether representing the sense or antisense strand.
  • nucleotide sequence in relation to the present invention includes genomic DNA, cDNA, synthetic DNA, and RNA. Preferably it means DNA, more preferably cDNA for the coding sequence of the present invention.
  • the nucleotide sequence per se of the present invention does not cover the native nucleotide sequence according to the present invention in its natural environment when it is linked to its naturally associated sequence(s) that is/are also in its/their natural environment.
  • the term "non-native nucleotide sequence" means an entire nucleotide sequence that is in its native environment and when operatively linked to an entire promoter with which it is naturally associated, which promoter is also in its native environment.
  • the amino acid sequence of the present invention can be isolated and/or purified post expression of a nucleotide sequence in its native organism.
  • the amino acid sequence of the present invention may be expressed by a nucleotide sequence in its native organism but wherein the nucleotide sequence is not under the control of the promoter with which it is naturally associated within that organism.
  • the nucleotide sequence of the present invention is prepared using recombinant DNA techniques (i.e. recombinant DNA).
  • the nucleotide sequence could be synthesised, in whole or in part, using chemical methods well known in the art (see Caruthers MH et al (1980) Nuc Acids Res Symp Ser 215-23 and Horn T et al (1980) Nuc Acids Res Symp Ser 225-232).
  • a nucleotide sequence encoding either an enzyme which has the specific properties as defined herein or an enzyme which is suitable for modification may be identified and/or isolated and/or purified from any cell or organism producing said enzyme.
  • Various methods are well known within the art for the identification and/or isolation and/or purification of nucleotide sequences. By way of example, PCR amplification techniques to prepare more of a sequence may be used once a suitable sequence has been identified and/or isolated and/or purified.
  • a genomic DNA and/or cDNA library may be constructed using chromosomal DNA or messenger RNA from the organism producing the enzyme. If the amino acid sequence of the enzyme is known, labelled oligonucleotide probes may be synthesised and used to identify enzyme-encoding clones from the genomic library prepared from the organism. Alternatively, a labelled oligonucleotide probe containing sequences homologous to another known enzyme gene could be used to identify enzyme-encoding clones. In the latter case, hybridisation and washing conditions of lower stringency are used.
  • enzyme-encoding clones could be identified by inserting fragments of genomic DNA into an expression vector, such as a plasmid, transforming enzyme- negative bacteria with the resulting genomic DNA library, and then plating the transformed bacteria onto agar containing a substrate for enzyme (i.e. maltose), thereby allowing clones expressing the enzyme to be identified.
  • an expression vector such as a plasmid
  • transforming enzyme- negative bacteria with the resulting genomic DNA library
  • the nucleotide sequence encoding the enzyme may be prepared synthetically by established standard methods, e.g. the phosphoroamidite method described by Beucage S.L. et al (1981) Tetrahedron Letters 22, p 1859-1869, or the method described by Matthes et al (1984) EMBO J. 3, p 801-805.
  • the phosphoroamidite method oligonucleotides are synthesised, e.g. in an automatic DNA synthesiser, purified, annealed, ligated and cloned in appropriate vectors.
  • the nucleotide sequence may be of mixed genomic and synthetic origin, mixed synthetic and cDNA origin, or mixed genomic and cDNA origin, prepared by ligating fragments of synthetic, genomic or cDNA origin (as appropriate) in accordance with standard techniques. Each ligated fragment corresponds to various parts of the entire nucleotide sequence.
  • the DNA sequence may also be prepared by polymerase chain reaction (PCR) using specific primers, for instance as described in US 4,683,202 or in Saiki R K et al (Science (1988) 239, pp 487-491).
  • the present invention also encompasses amino acid sequences of enzymes having the specific properties as defined herein.
  • amino acid sequence is synonymous with the term “polypeptide” and/or the term “protein”. In some instances, the term “amino acid sequence” is synonymous with the term “peptide”. In some instances, the term “amino acid sequence” is synonymous with the term “enzyme”.
  • amino acid sequence may be prepared/isolated from a suitable source, or it may be made synthetically or it may be prepared by use of recombinant DNA techniques.
  • the enzyme of the present invention may be used in conjunction with other enzymes.
  • the present invention also covers a combination of enzymes wherein the combination comprises the enzyme of the present invention and another enzyme, which may be another enzyme according to the present invention. This aspect is discussed in a later section.
  • the enzyme is not a native enzyme.
  • native enzyme means an entire enzyme that is in its native environment and when it has been expressed by its native nucleotide sequence.
  • the present invention also encompasses the use of variants, homologues and derivatives of any amino acid sequence of an enzyme of the present invention or of any nucleotide sequence encoding such an enzyme.
  • the term “homologue” means an entity having a certain homology with the subject amino acid sequences and the subject nucleotide sequences.
  • the term “homology” can be equated with "identity”.
  • an homologous sequence is taken to include an amino acid sequence which may be at least 75, 80, 85 or 90% identical, preferably at least 95, 96, 97, 98 -or 99% identical to the subject sequencer"
  • the homologues will comprise the same active sites etc. as the subject amino acid sequence.
  • homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present invention it is preferred to express homology in terms of sequence identity.
  • an homologous sequence is taken to include a nucleotide sequence which may be at least 40, 50, 60, 70, 75, 80, 85 or 90% identical, preferably at least 95, 96, 97, 98 or 99% identical to a nucleotide sequence encoding an enzyme of the present invention (the subject sequence).
  • the homologues will comprise the same sequences that code for the active sites etc. as the subject sequence.
  • homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present invention it is preferred to express homology in terms of sequence identity.
  • Homology comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate % homology between two or more sequences.
  • % homology may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid in one sequence is directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues.
  • a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance.
  • An example of such a matrix commonly used is the BLOSUM62 matrix - the default matrix for the BLAST suite of programs.
  • GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied (see user manual for further details). For some applications, it is preferred to use the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62.
  • percentage homologies may be calculated using the multiple alignment feature in DNASISTM (Hitachi Software), based on an algorithm, analogous to CLUSTAL (Higgins DG & Sharp PM (1988), Gene 73(1), 237-244).
  • % homology preferably % sequence identity.
  • the software typically does this as part of the sequence comparison and generates a numerical result.
  • sequences may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent substance.
  • Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues as long as the secondary binding activity of the substance is retained.
  • negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.
  • the present invention also encompasses homologous substitution (substitution and replacement are both used herein to mean the interchange of an existing amino acid residue, with an alternative residue) that may occur i.e. like-for-like substitution such as basic for basic, acidic for acidic, polar for polar etc.
  • Non-homologous substitution may also occur i.e.
  • Z ornithine
  • B diaminobutyric acid ornithine
  • O norleucine ornithine
  • pyriylalanine thienylalanine
  • naphthylalanine phenylglycine
  • Replacements may also be made by unnatural amino acids include; alpha* and alpha- disubstituted* amino acids, N-alkyl amino acids*, lactic acid*, halide derivatives of natural amino acids such as trifluorotyrosine*, p-Cl-phenylalanine*, p-Br- phenylalanine*, p-I-phenylalanine*, L-allyl-glycine*, ⁇ -alanine*, L- ⁇ -amino butyric acid*, L- ⁇ -amino butyric acid*, L- ⁇ -amino isobutyric acid*, L- ⁇ -amino caproic acid , 7-amino heptanoic acid*, L-methionine sulfone , L-norleucine*, L-norvaline*, p-nitro-
  • L-phenylalanine* L-hydroxyproline , L-thioproline*, methyl derivatives of phenylalanine (Phe) such as 4-methyl-Phe*, pentamethyl-Phe*, L-Phe (4-amino) , L-
  • Variant amino acid sequences may include suitable spacer groups that may be inserted between any two amino acid residues of the sequence including alkyl groups such as methyl, ethyl or propyl groups in addition to amino acid spacers such as glycine or ⁇ - alanine residues.
  • alkyl groups such as methyl, ethyl or propyl groups
  • amino acid spacers such as glycine or ⁇ - alanine residues.
  • a further form of variation involves the presence of one or more amino acid residues in peptoid form, will be well understood by those skilled in the art.
  • the peptoid form is used to refer to variant amino acid residues wherein the ⁇ -carbon substituent group is on the residue's nitrogen atom rather than the ⁇ -carbon.
  • Suitable fragments will be at least 5, e.g. 10, 12, 15 or 20 amino acids in length. They may also be less than 100, 75 or 50 amino acids in length. They may contain one or more (e.g. 5, 10, 15 or 20) substitutions, deletions or insertions, including conserved substitutions.
  • the nucleotide sequences for use in the present invention may include within them synthetic or modified nucleotides.
  • a number of different types of modification to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones and/or the addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule.
  • the nucleotide sequences described herein may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or life span of nucleotide sequences of the present invention.
  • the present invention also encompasses the use of nucleotide sequences that are complementary to the sequences presented herein, or any derivative, fragment or derivative thereof. If the sequence is complementary to a fragment thereof then that sequence can be used as a probe to identify similar coding sequences in other organisms etc.
  • Polynucleotides which are not 100% homologous to the sequences of the present invention but fall witMii the scope of the invention can be obtained in a number of ways.
  • Other variants of the sequences described herein may be obtained for example by probing DNA libraries made from a range of individuals, for example individuals from different populations.
  • other viral/bacterial, or cellular homologues particularly cellular homologues found in mammalian cells e.g. rat, mouse, bovine and primate cells
  • such homologues and fragments thereof in general will be capable of selectively hybridising to the sequences shown in the sequence listing herein.
  • sequences may be obtained by probing cDNA libraries made from or genomic DNA libraries from other animal species, and probing such libraries with probes comprising all or part of any one of the sequences in the attached sequence listings under conditions of medium to high stringency. Similar considerations apply to obtaining species homologues and allelic variants of the polypeptide or nucleotide sequences of the invention.
  • Variants and strain species homologues may also be obtained using degenerate PCR which will use primers designed to target sequences within the variants and homologues encoding conserved amino acid sequences within the sequences of the present invention.
  • conserveed sequences can be predicted, for example, by aligning the amino acid sequences from several variants/homologues. Sequence alignments can be performed using . computer software known in the art. For example the GCG Wisconsin PileUp program is widely used.
  • the primers used in degenerate PCR will contain one or more degenerate positions and will be used at stringency conditions lower than those used for cloning sequences with single sequence primers against known sequences.
  • polynucleotides may be obtained by -site directed mutagenesis of characterised sequences. This may be useful where for example silent codon sequence changes are required to optimise codon preferences for a particular host cell in which the polynucleotide sequences are being expressed. Other sequence changes may be desired in order to introduce restriction enzyme recognition sites, or to alter the property or function of the polypeptides encoded by the polynucleotides.
  • The- present, invention also encompasses polynucleotides which have undergone molecular evolution via random processes, selection mutagenesis or in vitro recombination.
  • polynucleotides which have undergone molecular evolution via random processes, selection mutagenesis or in vitro recombination.
  • mutations or natural variants of a polynucleotide sequence can be recombined with either the wildtype or other mutations or natural variants to produce new variants. Such new variants can also be screened for improved functionality of the encoded polypeptide.
  • optimised expression and/or activity in a host cell or in vitro include, but are not limited to one or more of the following: optimised expression and/or activity in a host cell or in vitro, increased enzymatic activity, altered substrate and/or product specificity, increased or decreased enzymatic or structural stability, altered enzymatic activity/specificity in preferred environmental conditions, e.g. temperature, pH, substrate.
  • Polynucleotides (nucleotide. sequences) of the invention may be used to produce a primer, e.g. a PCR primer, a primer for an alternative amplification reaction, a probe e.g. labelled with a revealing label by conventional means using radioactive or non-radioactive labels, or the polynucleotides may be cloned into vectors.
  • a primer e.g. a PCR primer, a primer for an alternative amplification reaction, a probe e.g. labelled with a revealing label by conventional means using radioactive or non-radioactive labels, or the polynucleotides may be cloned into vectors.
  • primers, probes and other fragments will be at least 15, preferably at least 20, for example at least 25, 30 or 40 nucleotides in length, and are also encompassed by the term polynucleotides of the invention as used herein.
  • Polynucleotides such as DNA polynucleotides and probes according to the invention may be produced recombinantly, synthetically, or by any means available to those of skill in the art. They may also be cloned by standard techniques.
  • primers will be produced by synthetic means, involving a stepwise manufacture of the desired nucleic acid sequence one nucleotide at a time. Techniques for accomplishing this using automated techniques are readily available in the art.
  • Longer polynucleotides will generally be produced using recombinant means, for example using a PCR (polymerase chain reaction) cloning techniques. This will involve making a pair of primers (e.g. of about 15 to 30 nucleotides) flanking a region of the lipid targeting sequence which it is desired to clone, bringing the primers into contact with mRNA or cDNA obtained from an animal or human cell, performing a polymerase chain reaction under conditions which bring about amplification of the desired region, isolating the amplified fragment (e.g. by purifying the reaction mixture on an agarose gel) and recovering the amplified DNA.
  • the primers may be designed to contain suitable restriction enzyme recognition sites so that the amplified DNA can be cloned into a suitable cloning vector. BIOLOGICALLY ACTIVE
  • the variant sequences etc. are at least as biologically active as the sequences presented herein.
  • biologically active refers to a sequence having a similar structural function (but not necessarily to the same degree), and/or similar regulatory function (but not necessarily to the same degree), and/or similar biochemical function (but not necessarily to the same degree) of the naturally occurring sequence.
  • the polypeptide of the present invention may exist in the form of one or more different isozymes.
  • isozyme encompasses variants of the polypeptide that catalyse the same reaction, but differ from each other in properties such as substrate affinity and maximum rates of enzyme-substrate reaction. Owing to differences in amino acid sequence, isozymes can be distinguished by techniques such as electrophoresis or isoelectric focusing. Different tissues often have different isoenzymes. The sequence differences generally confer different enzyme kinetic parameters that can sometimes be interpreted as fine tuning to the specific requirements of the cell types in which a particular isoenzyme is found.
  • the present invention also encompasses sequences that are complementary to the sequences of the present invention or sequences that are capable of hybridising either to the sequences of the present invention or to sequences that are complementary thereto.
  • hybridisation shall include “the process by which a strand of nucleic acid joins with a complementary strand through base pairing” as well as the process of amplification as carried out in polymerase chain reaction (PCR) technologies.
  • PCR polymerase chain reaction
  • the present invention also encompasses the use of nucleotide sequences that are capable of hybridising to the sequences that are complementary to the sequences presented herein, or any derivative, fragment or derivative thereof.
  • variant also encompasses sequences that are complementary to sequences that are capable of hybridising to the nucleotide sequences presented herein.
  • the present invention also relates to nucleotide sequences that can hybridise to the nucleotide sequences of the present invention (including complementary sequences of those presented herein).
  • the present invention also relates to nucleotide sequences that are complementary to sequences that can hybridise to the nucleotide sequences of the present invention (including complementary sequences of those presented herein).
  • polynucleotide sequences that are capable of hybridising to the nucleotide sequences presented herein under conditions of intermediate to maximal stringency.
  • the present invention covers nucleotide sequences that can hybridise to the nucleotide sequence of the present invention; or the complement thereof, under stringent conditions (e.g. 50°C and 0.2xSSC). In a more preferred aspect, the present invention covers nucleotide sequences that can hybridise to the nucleotide sequence of the present invention, or the complement thereof, under high stringent conditions (e.g. 65°C and O.lxSSC).
  • stringent conditions e.g. 50°C and 0.2xSSC
  • high stringent conditions e.g. 65°C and O.lxSSC
  • an enzyme-encoding nucleotide sequence has been isolated, or a putative enzyme-encoding nucleotide sequence has been identified, it may be desirable to mutate the sequence in order to prepare an enzyme of the present invention.
  • Mutations may be. introduced using synthetic oligonucleotides. " These oli ' gohucleoticles contain nucleotide sequences flanking the desired mutation sites.
  • a suitable method is disclosed in Morinaga et al (Biotechnology (1984) 2, p646-649), wherein a single-stranded gap of DNA, the enzyme-encoding sequence, is created in a vector carrying the enzyme gene.
  • the synthetic nucleotide, bearing the desired mutation, is then annealed to a homologous portion of the single-stranded DNA.
  • the remaining gap is then filled in with DNA polymerase I (Klenow fragment) and the construct is ligated using T4 ligase.
  • Sierks et al (Protein Eng (1989) 2, 621-625 and Protein Eng (1990) 3, 193-198) describes site-directed mutagenesis Aspergillus glucoamylase.
  • the sequence is a recombinant sequence - i.e. a sequence that has been prepared using recombinant DNA techniques.
  • the sequence is a synthetic sequence - i.e. a sequence that has been prepared by in vitro chemical or enzymatic synthesis. It includes but is not limited to sequences made with optimal codon usage for host organisms, such as the methylotrophic yeasts Pichia and Hansenula.
  • the nucleotide sequence for use in the present invention can be incorporated into a recombinant replicable vector.
  • the vector may be used to replicate and express the nucleotide sequence, in enzyme form, in and/or from a compatible host cell. Both homologous and heterologous expression is contemplated.
  • the gene of interest or nucleotide sequence of interest is not in its naturally occurring genetic context.
  • expression is driven by means other than or in addition to its naturally occurring expression mechanism; for example, by overexpressing the gene of interest by genetic intervention.
  • Expression may be controlled using control sequences which include promoters/enhancers and other expression regulation signals.
  • Prokaryotic promoters and promoters functional in eukaryotic cells may be used.
  • Tissue specific or stimuli specific promoters may be used.
  • Chimeric promoters may also be used comprising sequence elements from two or more different promoters described above.
  • the enzyme produced by a host recombinant cell by expression of the nucleotide sequence may be secreted or may be contained intracellularly depending on the sequence and/or the vector used.
  • the coding sequences can be designed with signal sequences which direct secretion of the substance coding sequences through a particular prokaryotic or eukaryotic cell membrane.
  • expression vector means a construct capable of in vivo or in vitro expression.
  • the expression vector is incorporated in the genome of a suitable host organism.
  • the term "incorporated” preferably covers stable incorporation into the genome.
  • the host organism can be the same or different to the gene of interest source organism, giving rise to homologous and heterologous expression respectively.
  • the vector of the present invention comprises a construct according to the present invention.
  • the nucleotide sequence of the present invention is present in a vector and wherein the nucleotide sequence is operably linked to regulatory sequences such that the regulatory" sequences are capable of providing the expression of the nucleotide sequence by a suitable host organism, i.e. the vector is an expression vector.
  • the vectors of the present invention may be transformed into a suitable host cell as described below to provide for expression of a polypeptide of the present invention.
  • the invention provides a process for prepjaring polypeptides for subsequent use according to the present invention which comprises cultivating a host cell transformed or transfected with an expression vector under conditions to provide for expression by the vector of a coding sequence encoding the polypeptides, and recovering the expressed polypeptides.
  • the vectors may be for example, plasmid, virus or phage vectors provided with an origin of replication, optionally a promoter for the expression of the said polynucleotide and optionally a regulator of the promoter.
  • the choice of vector will often depend on the host cell into which it is to be introduced.
  • the vectors of the present invention may contain one or more selectable marker genes.
  • the most suitable selection systems for industrial micro-organisms are those formed by the group of selection markers which do not require a mutation in the host organism.
  • Suitable selection markers may be the dal genes from B. subtilis or B. licheniformis, or one which confers antibiotic resistance such as ampicillin, kanamycin, chloramphenicol or tetracyclin resistance.
  • Alternative selection markers may be the Aspergillus selection markers such as amdS, argB, niaD and sC, or a marker giving rise to hygromycin resistance.
  • Examples of other fungal selection markers are the genes for ATP synthetase, subunit 9 (oliC), orotidine-5'-phosphate-decarboxylase (pvrA), phleomycin and benomyl resistance (benA).
  • Examples of non-fungal selection markers are the bacterial G418 resistance gene (this may also be used in yeast, but not in filamentous fungi), the ampicillin resistance gene (E. coli), the neomycin resistance gene (Bacillus) and the E. coli uidh gene, coding for ⁇ -glucuronidase (GUS).
  • Further suitable selection markers include the dal genes from B subtilis or B. licheniformis. Alternatively, the selection may be accomplished by co-transformation (as described in WO91/17243).
  • Vectors may be used in vitro, for example for the production of RNA or used to transfect or transform a host cell.
  • nucleotide sequences for use according to the present invention can be incorporated into a recombinant vector (typically a replicable vector), for example a cloning or expression vector.
  • the vector may be used to replicate the nucleic acid in a compatible host cell.
  • the invention provides a method of making nucleotide sequences of the present invention by -introducing a nucleotide sequence of the present invention into a replicable vector, introducing the vector into a compatible host cell, and growing the host cell under conditions which bring about replication of the vector.
  • the vector may be recovered from the host cell. Suitable host cells are described below in connection with expression vectors.
  • the vector may further comprise a nucleotide sequence enabling the vector to replicate in the host cell in question.
  • sequences are the origins of replication of plasmids pUC19, pACYC177, pUBl 10, pE194, pAMBl and pIJ702.
  • the expression vector typically includes the components of a cloning vector, such as, for example, an element that permits autonomous replication of the vector in the selected host organism and one or more phenotypically detectable markers for selection purposes.
  • the expression vector normally comprises control nucleotide sequences encoding a promoter, operator, ribosome binding site, translation initiation signal and optionally, a repressor gene or one or more activator genes.
  • the expression vector may comprise a sequence coding for an amino acid sequence capable of targeting the amino acid sequence to a host cell organelle such as a peroxisome or to a particular host cell compartment.
  • the term 'expression signal includes any of the above control sequences, repressor or activator sequences.
  • the nucleotide sequence is operably linked to the control sequences in proper manner with respect to expression.
  • the nucleotide sequence for use in the present invention is operably linked to a regulatory sequence which is capable of providing for the expression of the nucleotide sequence, such as by the chosen host cell.
  • the present invention covers a vector comprising the nucleotide sequence of the present invention operably linked to such a regulatory sequence, i.e. the vector is an expression vector.
  • operably linked refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their-ihtended manner.
  • a regulatory sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under condition compatible with the control sequences.
  • regulatory sequences includes promoters and enhancers and other expression regulation signals.
  • promoter is used in the normal sense of the art, e.g. an RNA polymerase binding site.
  • Enhanced expression of the nucleotide sequence encoding the enzyme of the present invention may also be achieved by the selection of heterologous regulatory regions, e.g. promoter, secretion leader and terminator regions, which serve to increase expression and, if desired, secretion levels of the protein of interest from the chosen expression host and/or to provide for the inducible control of the expression of the enzyme of the present invention.
  • heterologous regulatory regions e.g. promoter, secretion leader and terminator regions
  • polyadenylation sequences may be operably connected to the nucleotide sequence encoding the enzyme.
  • the nucleotide sequence of the present invention may be operably linked to at least a promoter.
  • a promoter Aside from the promoter native to the gene encoding the nucleotide sequence of the present invention, other promoters may be used to direct expression of the polypeptide of the present invention.
  • the promoter may be selected for its efficiency in directing the expression of the nucleotide sequence of the present invention in the desired expression host.
  • a constitutive promoter may be selected to direct the expression of the desired nucleotide sequence of the present invention.
  • Such an expression construct may provide additional advantages since it circumvents the need to culture the expression hosts on a medium containing an inducing substrate.
  • suitable promoters for directing the transcription of the nucleotide sequence in a bacterial host include the promoter of the lac operon of E. coli, 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 amyloliquefaciens a- amylase gene (amyQ), the promoters of the Bacillus subtilis xylA ⁇ ndxylB genes and a promoter derived from a Lactococcus sp.-derived promoter including the P170 promoter.
  • a suitable promoter can be selected, for example, from a bacteriophage promoter including a T7 promoter and a phage lambda promoter.
  • examples of useful promoters are those derived from the genes encoding the, Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral ⁇ -amylase, A. niger acid stable ⁇ - amylase, A. niger glucoamylase, Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase or Aspergillus nidulans acetamidase.
  • strong constitutive and/or inducible promoters which are preferred for use in fungal expression hosts are those which are obtainable from the fungal genes for xylanase (xlnA), phytase, ATP-synthetase, subunit 9 (oliC), triose phosphate isomerase (tpi), alcohol dehydrogenase (AdhA), ⁇ -amylase (amy), amyloglucosidase (AG - from the glaA gene), acetamidase (amdS) and glyceraldehyde-3 -phosphate dehydrogenase
  • gpd gpd promoters.
  • TPI triose phosphate isomerase
  • S. cerevisiae the gene encoding A. oryzae TAKA amylase
  • TPI triose phosphate isomerase
  • 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.
  • Suitable promoters for the expression in a yeast species mciu ⁇ e out are not limited to the Gal 1 and Gal 10 promoters of Saccharomyces cerevisiae and the Pichia pastoris AOX1 or AOX2 promoters.
  • Hybrid promoters may also be used to improve inducible regulation of the expression construct.
  • the promoter can additionally include features to ensure or to increase expression in a suitable host.
  • the features can be conserved regions such as a Pribnow Box or a TATA box.
  • the promoter may even contain other sequences to affect (such as to maintain, enhance, decrease) the levels of expression of the nucleotide sequence of the present invention.
  • suitable other sequences include the Shl-intron or an ADH intron.
  • Other sequences include inducible elements - such as temperature, chemical, light or stress inducible elements.
  • suitable elements to enhance transcription or translation may be present. An example of the latter element is the TMV 5' signal sequence (see Sleat 1987 Gene 217, 217-225 and Dawson 1993 Plant Mol. Biol. 23: 97).
  • construct which is synonymous with terms such as “conjugate”, “cassette” and “hybrid” - includes a nucleotide sequence for use according to the present invention directly or indirectly attached to a promoter.
  • An example of an indirect attachment is the provision of a suitable spacer group such as an intron sequence, such as the Shl-intron or the ADH intron, intermediate the promoter and the nucleotide sequence of the present invention.
  • intron sequence such as the Shl-intron or the ADH intron
  • fused in relation to the present invention which includes direct or indirect attachment. In some cases, the terms do not cover the natural combination of the nucleotide sequence coding for the protein ordinarily associated with the wild type gene promoter and when they are both in their natural environment.
  • the construct may even contain or express a marker which allows for the selection of the genetic construct in, for example, a bacterium, preferably of the genus Bacillus, such as Bacillus subtilis, or plants into which it has been transferred.
  • a marker which allows for the selection of the genetic construct in, for example, a bacterium, preferably of the genus Bacillus, such as Bacillus subtilis, or plants into which it has been transferred.
  • markers which may be used, such as for example those encoding mannose-6-phosphate isomerase (especially for plants) or those markers that provide for antibiotic resistance - e.g. resistance to G418, hygromycin, bleomycin, kanamycin and gentamycin.
  • the construct of the present invention comprises at least the nucleotide sequence of the present invention operably linked to a promoter.
  • host cell in relation to the present invention includes any cell that comprises either the nucleotide sequence or an expression vector as described above and which is used in the recombinant production of an enzyme having the specific properties as defined herein.
  • the nucleotide of interest may be homologous or heterologous to the host cell.
  • a further embodiment of the present invention provides host cells transformed or transfected with a nucleotide sequence that expresses the enzyme of the present invention.
  • a nucleotide sequence is carried in a vector for the replication and expression of the nucleotide sequence.
  • the cells will be chosen to be compatible with the said vector and may for example be prokaryotic (for example bacterial), fungal, yeast or plant cells.
  • suitable bacterial host organisms are gram positive bacterial species such as Bacillaceae including Bacillus subtilis, Bacillus licheniformis, Bacillus lentus,
  • Bacillus brevis Bacillus stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus lautus, Bacillus megaterium and Bacillus thuringiensis, Streptomyces species such as Streptomyces murinus, lactic acid bacterial species including Lactococcus spp. such as Lactococcus lactis, Lactobacillus spp. including Lactobacillus reuteri, Leuconostoc spp., Pediococcus spp. and Streptococcus spp.
  • strains of a gram-negative bacterial species belonging to Enterobacteriace&Q including E. coli, or to Pseudomonadaceae can be selected as the host organism.
  • the gram negative bacterium E. coli is widely used as a host for heterologous gene expression.
  • large amounts of heterologous protein tend to accumulate inside the cell.
  • Subsequent purification of the desired protein from the bulk of E. coli intracellular proteins can sometimes be difficult.
  • Gram positive bacteria from the genus Bacillus such as B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. coagulans, B. circulans, B. lautus, B. megaterium, B. thuringiensis, Streptomyces lividans or S. murinus
  • Bacillus such as B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. coagulans, B. circulans, B. lautus, B. megaterium, B. thuringiensis, Streptomyces lividans or S. murinus
  • Other bacteria that may be suitable as hosts are those from the genera Streptomyces and Pseudomonas.
  • eukaryotic hosts such as yeasts or other fungi may be preferred.
  • yeast cells are preferred over fungal cells because they are easier to manipulate.
  • some proteins are either poorly secreted from the yeast cell, or in some cases are not processed properly (e.g. hyperglycosylation in yeast).
  • Typical fungal expression hosts may be selected from Aspergillus niger, Aspergillus niger var. tubigenis, Aspergillus niger var. awamori, Aspergillus aculeatis,
  • Bacillus licheniformis Bacillus amyloliquefaciens, Kluyveromyces lactis- and Saccharomyces cerevisiae.
  • Suitable filamentous fungus may be for example a strain belonging to a species of Aspergillus, such as Aspergillus oryzae or Aspergillus niger, or a strain of Fusarium oxysporium, Fusarium graminearum (in the perfect state named Gribberella zeae, previously Sphaeria zeae, synonym with Gibberella roseum and Gibberella roseum f. sp.
  • Fusaisum sulphureum in the perfect state named Gibberella puricaris, synonym with Fusarium trichothercioides, Fusarium bactridioides, Fusarium sambucium, Fusarium roseum and Fusarium roseum var. graminearum), Fusarium cerealis (synonym with Fusarium crokkwellnse) or Fusarium venenatum.
  • Suitable yeast organisms may be selected from the species of Kluyveromyces, Saccharomyces or Schizosaccharomyces, e.g. Saccharomyces cerevisiae, or Hansenula (disclosed in UK Patent Application No. 9927801.2).
  • suitable host cells - such as yeast, fungal and plant host cells - may provide for post-translational modifications (e.g. myristoylation, glycosylation, truncation, lapidation and tyrosine, serine or threonine phosphorylation) as may be needed to confer optimal biological activity on recombinant expression products of the present invention.
  • post-translational modifications e.g. myristoylation, glycosylation, truncation, lapidation and tyrosine, serine or threonine phosphorylation
  • the host cell may be a protease deficient or protease minus strain.
  • This may for example be the protease deficient strain Aspergillus oryzae JaL 125 having the alkaline protease gene named "alp" deleted. This strain is described in WO97/35956.
  • organism in relation to the present invention includes any organism that could comprise the nucleotide sequence coding for the enzyme according to the present invention and/or products obtained therefrom, and/or wherein a promoter can allow expression of the nucleotide sequence according to the present invention when present in the organism.
  • Suitable organisms may include a prokaryote, fungus, yeast or a plant.
  • transgenic organism in relation to the present invention includes any organism that comprises the nucleotide sequence coding for the enzyme according to the present invention and/or the products obtained therefrom, and/or wherein a promoter can allow expression of the nucleotide sequence according to the present invention within the " organism.
  • the nucleotide sequence is incorporated in the genome of the organism.
  • transgenic organism does not cover native nucleotide coding sequences in their natural environment when they are under the control of their native promoter which is also in its natural environment.
  • the transgenic organism of the present invention includes an organism comprising any one of, or combinations of, the nucleotide sequence coding for the enzyme according to the present invention, constructs according to the present invention, vectors according to the present invention, plasmids according to the present invention, cells according to the present invention, tissues according to the present invention, or the products thereof.
  • the transgenic organism can also comprise the nucleotide sequence coding for the enzyme of the present invention under the control of a heterologous promoter.
  • the host organism can be a prokaryotic or a eukaryotic organism.
  • suitable prokaryotic hosts include E. coli and Bacillus subtilis. Teachings on the transformation of prokaryotic hosts is well documented in the art, for example see Sambrook et al (Molecular Cloning: A Laboratory Manual, 2nd edition,
  • nucleotide sequence may need to be suitably modified before transformation - such as by removal of introns.
  • Filamentous fungi 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.
  • the use oi Aspergillus as a host microorganism is described in EP 0238 023.
  • Another host organism can be a plant.
  • the basic principle in the construction of genetically modified plants is to insert genetic information in the plant genome so as to obtain a stable maintenance of the inserted genetic material.
  • Several techniques exist for inserting the genetic information the two main principles being direct introduction of the genetic information and introduction of the genetic information by use of a vector system.
  • a review of the general techniques may be found in articles by Potrykus (Annu Rev Plant Physiol Plant Mol Biol [1991] 42:205-225) and Christou (Agro-Food-Industry Hi-Tech March April 1994 17-27). Further teachings on plant transformation may be found in EP-A-0449375.
  • a host organism may be a fungus - such as a mold.
  • suitable such hosts include any member belonging to the genera Thermomyces, Acremonium, Aspergillus, Penicillium, Mucor, Neurospora, Trichoderma and the like - such as Thermomyces lanuginosis, Acremonium chrysogenum, Aspergillus niger, Aspergillus oryzae, Aspergillus awamori, Penicillinum chrysogenem, Mucor javanious, Neurospora crassa, Trichoderma viridae and the like.
  • the host organism may be a filamentous fungus.
  • filamentous fungi have been widely used in many types of industry for the production of organic compounds and enzymes.
  • traditional Japanese koji and soy fermentations have used Aspergillus sp.
  • Aspergillus niger has been used for production of organic acids particular citric acid and for production of various enzymes for use in industry.
  • filamentous fungi There are two major reasons why filamentous fungi have been so widely used in industry. First filamentous fungi can produce high amounts of extracellular products, for example enzymes and organic compounds such as antibiotics or orgaMfacids. Second filamentous fungi can grow on low cost substrates such as grains, bran, beet pulp etc. The same reasons have made filamentous fungi attractive organisms as hosts for heterologous expression according to the present invention.
  • expression constructs are prepared by inserting the nucleotide sequence according to the present invention into a construct designed for expression in filamentous fungi.
  • constructs used for heterologous expression preferably contain one or more of: a signal sequence which directs the amino acid sequence to be secreted, typically being of fungal origin, and a terminator (typically being active in fungi) which ends the expression system.
  • nucleotide sequence according to the present invention can be fused to a smaller or a larger part of a fungal gene encoding a stable protein.
  • This can stabilise the amino acid sequence.
  • a cleavage site recognised by a specific protease, can be introduced between the fungal protein and the amino acid sequence, so the produced fusion protein can be cleaved at this position by the specific protease thus liberating the amino acid sequence.
  • a site which is recognised by a KEX-2 like peptidase found in at least some Aspergilli Such a fusion leads to cleavage in vivo resulting in production of the expressed product and not a larger fusion protein.
  • Heterologous expression in Aspergillus has been reported for several genes coding for bacterial, fungal, vertebrate and plant proteins.
  • the proteins can be deposited intracellularly if the nucleotide sequence according to the present invention is not fused to a signal sequence. Such proteins will accumulate in the cytoplasm and will usually not be glycosylated which can be an advantage for some bacterial proteins. If the nucleotide sequence according to the present invention is equipped with a signal sequence the protein will accumulate extracellularly.
  • heterologous proteins are not very stable when they are secreted into the culture fluid of fungi. Most fungi produce several extracellular proteases which degrade heterologous proteins. To avoid this problem special fungal strains with reduced protease production have been used as host for heterologous production.
  • Host strains can generally be grown in either Nogel's or Fries minimal medium supplemented with the appropriate nutrient(s), such as, for example, any one or more of: his, arg, phe, tyr, trp, p-aminobenzoic acid, and inositol.
  • appropriate nutrient(s) such as, for example, any one or more of: his, arg, phe, tyr, trp, p-aminobenzoic acid, and inositol.
  • the transformed protoplasts regenerate and the transformed fungi are selected using various selective markers.
  • the transformation typically also involves a selectable gene marker which is introduced with the expression cassette, either on the same vector or by co-transformation, into a host strain in which the gene marker is selectable.
  • a selectable gene marker which is introduced with the expression cassette, either on the same vector or by co-transformation, into a host strain in which the gene marker is selectable.
  • Various marker/host systems are available, including the pyrG, argB and niaD genes for use with auxotrophic strains of Aspergillus nidulans; pyrG and argB genes for Aspergillus oryzae-auxotrophs; pyrG, t C ⁇ and niaD genes for Penicillium chrysogenum auxotrophs; and the argB gene for Trichoderma reesei auxotrophs.
  • Dominant selectable markers including amdS, oliC, hyg and phleo are also now available for use with such filamentous fungi as A. niger, A. oryzae, A. flcuum, P. chrysogenum, Cephalosporium acremonium, Cochliobolus heterostrophus, Glomerella cingulata, Fulviafulva and Leptosphaeria maculans (for a review see Ward in Modern Microbial Genetics, 1991, Wiley-Liss, Inc., at pages 455-495).
  • a commonly used transformation marker is the amdS gene of A. nidulans which in high copy number allows the fungus to grow with acrylamide as the sole nitrogen source.
  • auxotrophic markers such as argB, trpC, niaD and pyrG
  • antibiotic resistance markers such as benomyl resistance, hygromycin resistance and phleomycin resistance.
  • the host organism can be of the genus Aspergillus, such as Aspergillus niger.
  • a transgenic Aspergillus according to the present invention can also be prepared by following the teachings of Rambosek, J. and Leach, J. 1987 (Recombinant DNA in filamentous fungi: Progress and Prospects. CRC Crit. Rev. Biotechnol. 6:357-393),
  • the transgenic organism can be a yeast.
  • yeast have also been widely used as a vehicle for heterologous gene expression.
  • Saccharomyces cerevisiae has a long history of industrial use, including its use for heterologous gene expression.
  • Expression of heterologous genes in Saccharomyces cerevisiae has been reviewed by Goodey et al (1987, Yeast Biotechnology, D R Berry et al, eds, pp 401-429, Allen and Unwin, London) and by King et al (1989, Molecular and Cell Biology of Yeasts, E F Walton and G T Yarronton, eds, pp 107-133, Blackie, Glasgow).
  • Saccharomyces cerevisiae is well suited for heterologous gene expression. First, it is non-pathogenic to humans and it is incapable of producing certain endotoxins. Second, it has a long history of safe use following centuries of commercial exploitation for various purposes. This has led to wide public acceptability. Third, the extensive commercial use and research devoted to the organism has resulted in a wealth of knowledge about the genetics and physiology as well as large-scale fermentation characteristics of Saccharomyces cerevisiae.
  • yeast vectors include integrative vectors, which require recombination with the host genome for their maintenance, and autonomously replicating plasmid vectors.
  • expression constructs are prepared by inserting the nucleotide sequence of the present invention into a construct designed for expression in yeast.
  • the constructs may contain a promoter active in yeast, such as a promoter of yeast origin, such as the GAL1 promoter, is used.
  • a promoter of yeast origin such as the GAL1 promoter
  • a signal sequence of yeast origin such as the sequence encoding the SUC2 signal peptide
  • a terminator active in yeast ends the expression system.
  • transgenic Saccharomyces can be prepared by following the teachings of Hinnen et al (1978, Proceedings of the National Academy of Sciences of the USA 75, 1929); Beggs, J D (1978, Nature, London, 275, 104); and Ito, H et al (1983, J Bacteriology 153, 163-168).
  • the transformed yeast cells may be selected using various selective markers.
  • markers used for transformation are a number of auxotrophic markers such as LEU2, HIS4 and TRPl, and dominant antibiotic resistance markers such as aminoglycoside antibiotic markers, eg G418.
  • a preferred host organism suitable for the present invention is a plant.
  • the basic principle in the construction of genetically modified plants is to insert genetic information in the plant genome so as to obtain a stable maintenance of the inserted genetic material.
  • the basic principle in the construction of genetically modified plants is to insert genetic information in the plant genome so as to obtain a stable maintenance of the inserted genetic material.
  • the present invention relates to a vector system which carries a nucleotide sequence or construct according to the present invention and which is capable of introducing the nucleotide sequence or construct into the genome of an organism, such as a plant.
  • the vector system may comprise one vector, but it can comprise two vectors. In the case of two vectors, the vector system is normally referred to as a binary vector system.
  • Binary vector systems are described in further detail in Gynheung An et al. (1980), Binary Vectors, Plant Molecular Biology Manual A3, 1-19.
  • Ti and Ri plasmids have been constructed which are suitable for the construction of the plant or plant cell constructs described above.
  • a non-limiting example of such a Ti plasmid is pGV3850.
  • the nucleotide sequence or construct of the present invention should preferably be inserted into the Ti-plasmid between the terminal sequences of the T-DNA or adjacent a T-DNA sequence so as to avoid disruption of the sequences immediately surrounding the T-DNAsborders, as at least one "of these regions appear to be essential for insertion ot modified T-DNA into the plant genome.
  • the vector system of the present invention is preferably one which contains the sequences necessary to infect the plant (e.g. the vir region) and at least one border part of a T-DNA sequence, the border part being located on the same vector as the genetic construct.
  • the vector system is an Agrobacterium tumefaciens Ti-plasmid or an Agrobacterium rhizogenes Ri-plasmid or a derivative thereof, as these plasmids are well- known and widely employed in the construction of transgenic plants, many vector systems exist which are based on these plasmids or derivatives thereof.
  • the nucleotide sequence or construct of the present invention may be first constructed in a micro-organism in which the vector can replicate and which is easy to manipulate before insertion into the plant.
  • An example of a useful micro-organism is E. coli, but other micro-organisms having the above properties may be used.
  • a vector of a vector system as defined above has been constructed in E. coli. it is transferred, if necessary, into a suitable Agrobacterium strain, e.g. Agrobacterium tumefaciens.
  • the Ti-plasmid harbouring the nucleotide sequence or construct of the invention is thus preferably transferred into a suitable Agrobacterium strain, e.g. A. tumefaciens, so as to obtain an Agrobacterium cell harbouring the nucleotide sequence or construct of the invention, which DNA is subsequently transferred into the plant cell to be modified.
  • cloning vectors which contain a replication system in E. coll and. a -marker which allows a selection of the transformed cells.
  • the vectors contain for example pBR 322, the pUC series, the Ml 3 mp series, pACYC 184 etc.
  • the nucleotide or construct of the present invention can be introduced into a suitable restriction position in the vector.
  • the contained plasmid is used for the transformation in E.coli.
  • the E.coli cells are cultivated in a suitable nutrient medium and
  • plasmid is then recovered: As ⁇ a method of analysis"ffiere is generally used sequence analysis, restriction analysis, electrophoresis and further biochemical-molecular biological methods. After each manipulation, the used DNA sequence can be restricted and connected with the next DNA sequence. Each sequence can be cloned in the same or different plasmid.
  • the presence and/or insertion of further DNA sequences may be necessary. If, for example, for the transformation the Ti- or Ri-plasmid of the plant cells is used, at least the right boundary and often however the right and the left boundary of the Ti- and Ri-plasmid T-DNA, as flanking areas of the introduced genes, can be connected.
  • T-DNA for the transformation of plant cells has been intensively studied and is described in EP-A-120516; Hoekema, in: The Binary Plant Vector System Offset-drukkerij Kanters B.B., Alblasserdam, 1985, Chapter V; Fraley, et ah, Crit. Rev. Plant Sci., 4:1-46; and An et al, EMBO J. (1985) 4:277-284.
  • a plant to be infected is wounded, e.g. by cutting the plant with a razor or puncturing the plant with a needle or rubbing the plant with an abrasive.
  • the wound is then inoculated with the Agrobacterium.
  • the inoculated plant or plant part is then grown on a suitable culture medium and allowed to develop into mature plants.
  • tissue culturing methods such as by culturing the cells in a suitable culture medium supplied with the necessary growth factors such as amino acids, plant hormones, vitamins, etc.
  • Regeneration of the transformed cells into genetically modified plants may be accomplished using known methods for the regeneration of plants from cell or tissue cultures, for example by selecting transformed shoots using an antibiotic and by subculturing the shoots on a medium containing the appropriate nutrients, plant hormones, etc.
  • transgenic plants are well known in the art.
  • either whole plants, cells or protoplasts may be transformed with a suitable nucleic acid construct encoding a zinc finger molecule or target DNA (see above for examples of nucleic acid constructs).
  • a suitable nucleic acid construct encoding a zinc finger molecule or target DNA (see above for examples of nucleic acid constructs).
  • Suitable methods include Agrobacterium infection (see, among others, Turpen et al, 1993, J. Virol. Methods, 42: 227-239) or direct delivery of DNA such as, for example, by PEG-mediated transformation, by electroporation or by acceleration of DNA coated particles. Acceleration methods are generally preferred and include, for example, microprojectile bombardment.
  • the ballistic transformation technique (otherwise known as the particle bombardment technique) was first described by Klein et al. [1987], Sanford et al. [1987] and -Klein et al. [1988] and has become widespread due to easy handling and the lack of pre-treatment of the cells or tissue in interest.
  • the principle of the particle bombardment technique is direct delivery of DNA-coated micro-projectiles into intact plant cells by a driving force (e.g. electrical discharge or compressed air).
  • a driving force e.g. electrical discharge or compressed air.
  • the micro-projectiles penetrate the cell wall and membrane, with only minor damage, and the transformed cells then express the promoter constructs.
  • PIG Particle Inflow Gun
  • the PIG accelerates the micro-projectiles in a stream of flowing helium, through a partial vacuum, into the plant cells.
  • One of advantages of the PIG is that the acceleration of the micro-projectiles can be controlled by a timer-relay solenoid and by regulation the provided helium pressure.
  • pressurised helium as a driving force has the advantage of being inert, leaves no residues and gives reproducible acceleration.
  • the vacuum reduces the drag on the particles and lessens tissue damage by dispersion of the helium gas prior to impact [Finer et al. 1992].
  • non-biological particles may be coated with nucleic acids and delivered into cells by a propelling force.
  • Exemplary particles include those comprised of tungsten, gold, platinum, and the like.
  • a particular advantage of microprojectile bombardment in addition to it being an effective means of reproducibly stably transforming both dicotyledons and monocotyledons, is that neither the isolation of protoplasts nor the susceptibility to Agrobacterium infection is required.
  • An illustrative embodiment of a method for delivering DNA into plant cells by acceleration is a Biolistics Particle Delivery System, which can be used to propel particles coated with DNA through a screen, such as a stainless steel or Nytex screen, onto a filter surface covered with plant cells cultured in suspension. The screen disperses the tungsten-DNA particles so that they are not delivered to the recipient cells in large aggregates.
  • the projectiles aggregate and may be too large for attaining a high frequency of transformation. This may be due to damage inflicted on the recipient cells by projectiles that are too large.
  • cells in suspension are preferably concentrated on filters. Filters containing the cells to be bombarded are positioned at an appropriate distance below the macroprojectile stopping plate. If desired, one or more screens are also positioned between the gun and the cells to be bombarded. Through the use of techniques set forth herein one may obtain up to 1000 or more clusters of cells transiently expressing a marker gene ("foci") on the bombarded filter.
  • the number of cells in a focus which express the exogenous gene product 48 hours post-bombardment often range from 1 to
  • a preferred step is to identity tne transrorme ⁇ DCis ior iuruier culturing and plant regeneration. This step may include assaying cultures directly for a screenable trait or by exposing the bombarded cultures to a selective agent or agents.
  • An example of a screenable marker trait is the red pigment produced under the control of the R-locus in maize.
  • This pigment may be detected by culturing cells on a solid support containing nutrient media capable of supporting growth at this stage, incubating the cells at, e.g., 18°C and greater than 180 ⁇ E m "2 s "1 , and selecting cells from colonies (visible aggregates of cells) that are pigmented. These cells may be cultured further, either in suspension or on solid media.
  • An exemplary embodiment of methods for identifying transformed cells involves exposing the bombarded cultures to a selective agent, such as a metabolic inhibitor, an antibiotic, herbicide or the like.
  • a selective agent such as a metabolic inhibitor, an antibiotic, herbicide or the like.
  • Cells which have been transformed and have stably integrated a marker gene conferring resistance to the selective agent used will grow and divide in culture. Sensitive cells will not be amenable to further culturing.
  • bombarded cells on filters are resuspended in nonselective liquid medium, cultured (e.g. for one to two weeks) and transferred to filters overlaying solid medium containing from 1-3 mg/1 bialaphos. While ranges of 1-3 mg/1 will typically be preferred, it is proposed that ranges of 0.1-50 mg/1 will find utility in the practice of the invention.
  • the type of filter for use in bombardment is not believed to be particularly crucial, and can comprise any solid, porous, inert support.
  • Cells that survive the exposure to the selective agent may be cultured in media that supports regeneration of plants. Tissue is maintained on a basic media with hormones for about 2-4 weeks, then transferred to media with no hormones. After 2-4 weeks, shoot development will signal the time to transfer to another media.
  • Regeneration typically requires a progression of media whose composition has been modified to provide the appropriate nutrients and hormonal signals during sequential developmental stages from the transformed callus to the more mature plant.
  • Developing plantlets are transferred to soil, and hardened, e.g., in an environmentally controlled chamber at about 85% relative humidity, 600 ppm CO 2 , and 250 ⁇ E m "2 s "1 of light.
  • Plants are preferably matured either in a growth chamber or greenhouse. Regeneration will typically take about 3-12 weeks.
  • cells are grown on solid media in tissue culture vessels.
  • An illustrative embodiment of such a vessel is a petri dish.
  • Regenerating plants are preferably grown at about 19°C to 28°C. After the regenerating plants have reached the stage of shoot and root development, they may be transferred to a greenhouse for further growth and testing.
  • Genomic DNA may be isolated from callus cell lines and plants to determine the presence of the exogenous gene through the use of techniques well known to those skilled in the art such as PCR and/or Southern blotting.
  • Host cells transformed with the nucleotide sequence may be cultured under conditions conducive to the production of the encoded enzyme and which facilitate recovery of the enzyme 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 questions and obtaining expression of the enzyme. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g. as described in catalogues of the American Type Culture Collection).
  • the protein produced by a recombinant cell may be displayed on the surface of the cell.
  • expression vectors containing coding sequences can be designed with signal sequences which direct secretion of the coding sequences through a particular prokaryotic or eukaryotic cell membrane.
  • Other recombinant constructions may join the coding sequence to nucleotide sequence encoding a polypeptide domain which will facilitate purification of soluble proteins (Kroll DJ et al (1993) DNA Cell Biol 12:441-53).
  • the enzyme may be secreted from the host cells and may conveniently be recovered from the culture medium by well-known procedures, including separating the cells from the medium by centrifugation or filtration, and precipitating proteinaceous components of the medium by means of a salt such as ammonium sulphate, followed by the use of chromatographic procedures such as ion exchange chromatography, affinity chromatography, or the like.
  • the secretion leader sequence may be selected on the basis of the desired expression host.
  • Hybrid signal sequences may also be used with the context of the present invention.
  • Typical examples of heterologous secretion leader sequences are those originating from the fungal amyloglucosidase (AG) gene (glaA - both 18 and 24 amino acid versions e.g. from Aspergillus), the a-factor gene (yeasts e.g. Saccharomyces, Kluyveromyces and Hansenula) or the ⁇ -amylase gene (Bacillus).
  • a variety of protocols for detecting and measuring the expression of the amino acid sequence are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescent activated cell sorting (FACS).
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • FACS fluorescent activated cell sorting
  • a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on the POI may be used or a competitive binding assay may be employed. These and other assays are described, among other places, in Hampton R et al (1990, Serological Methods, A Laboratory Manual, APS Press, St Paul MN) and Maddox DE et al (1983, J Exp Med 15 8:121 1).
  • Means for producing labelled hybridization or PCR probes for detecting the amino acid sequence include oligolabelling, nick translation, end-labelling or PCR amplification using a labelled nucleotide.
  • the NOI, or any portion of it may be cloned into a vector for the production of an mRNA probe.
  • Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by addition of an appropriate RNA polymerase such as T7, T3 or SP6 and labeled nucleotides.
  • reporter molecules or labels include those radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles and the like.
  • Patents teaching the use of such labels include US-A-3,817,837; US-A-3,850,752; US-A-3,939,350; US-A- 3,996,345; US-A-4,277,437; US-A-4,275,149 and US-A-4,366,241.
  • recombinant immunoglobuHns may be produced as shown in US-A-4,816,567.
  • Additional methods to quantitate the expression of the amino acid sequence include radiolabeling (Melby PC et al 1993 J Immunol Methods 159:235-44) or biotinylating (Duplaa C et al 1993 Anal Biochem 229-36) nucleotides, coamplification of a control nucleic acid, and standard curves onto which the experimental results are interpolated. Quantitation of multiple samples may be speeded up by running the assay in an ELISA format where the oligomer of interest is presented in various dilutions and a spectrophotometric or calorimetric response gives rapid quantitation.
  • marker gene expression suggests that the nucleotide sequence is also present, its presence and expression should be confirmed.
  • recombinant cells containing nucleotide sequences can be identified by the absence of marker gene function.
  • a marker gene can be placed in tandem with a nucleotide sequence under the control of the promoter of the present invention or an alternative promoter (preferably the same promoter of the present invention). Expression of the marker gene in response to induction or selection usually indicates expression of the amino acid sequence as well.
  • host cells which contain the nucleotide sequence may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridization and protein bioassay or immunoassay techniques which include membrane-based, solution-based, or chip- based technologies for the detection and/or quantification of the nucleic acid or protein.
  • the amino acid sequence of the present invention may be produced as a fusion protein, for example to aid in extraction and purification.
  • fusion protein partners include glutathione-S-transferase (GST), 6xHis, GAL4 (DNA binding and/or transcriptional activation domains) and ( ⁇ -galactosidase. It may also be convenient to include a proteolytic cleavage site between the fusion protein partner and the protein sequence of interest to allow removal of fusion protein sequences. Preferably the fusion protein will not hinder the activity of the protein sequence.
  • the fusion protein may comprise an antigen or an antigenic determinant fused to the substance of the present invention.
  • the fusion protein may be a non-naturally occurring fusion protein comprising a substance which may act as an adjuvant in the sense of providing a generalised stimulation of the immune system.
  • the antigen or antigenic determinant may be attached to either the amino or carboxy terminus of the substance.
  • the amino acid sequence may be ligated to a heterologous sequence to encode a fusion protein.
  • a heterologous sequence for example, for screening of peptide libraries for agents capable of affecting the substance activity, it may be useful to encode a chimeric substance expressing a heterologous epitope that is recognised by a commercially available antibody.
  • sequences of the present invention may be used in conjunction with one or more additional proteins of interest (POIs) or nucleotide sequences of interest (NOIs).
  • POIs proteins of interest
  • NOIs nucleotide sequences of interest
  • Non-limiting examples of POIs include: proteins or enzymes involved in starch metabolism, proteins or enzymes involved in glycogen metabolism, acetyl esterases, aminopeptidases, amylases, arabinases, arabinofuranosidases, carboxypeptidases, catalases, cellulases, chitinases, chymosin, cutinase, deoxyribonucleases, epimerases, esterases, ⁇ -galactosidases, ⁇ -galactosidases, ⁇ -glucanases, glucan lysases, endo- ⁇ - glucanases, glucoamylases, glucose oxidases, ⁇ -glucosidases, ⁇ -glucosidases, glucuronidases, hemicellulases, hexose oxidases, hydrolases, invertases, isomerases, laccases, Upases,
  • NOI may even be an antisense sequence for any of " those sequences.
  • the POI may even be a fusion protein, for example to aid in extraction and purification.
  • fusion protein partners include the maltose binding protein, glutathione-S- transferase (GST), 6xHis, GAL4 (DNA binding and/or transcriptional activation domains) and ⁇ -galactosidase. It may also be convenient " to include a proteolytic cleavage site between the fusion components.
  • the POI may even be fused to a secretion sequence.
  • secretion leader sequences are those originating from the amyloglucosidase gene, the ⁇ -factor gene, the ⁇ -amylase gene, the lipase A gene, the xylanase A gene.
  • sequences can also facilitate secretion or increase the yield of secreted POI.
  • sequences could code for chaperone proteins as for example the product of Aspergillus niger cyp B gene described in UK patent application 9821198.0.
  • the NOI may be engineered in order to alter their activity for a number of reasons, including but not limited to, alterations which modify the processing and/or expression of the expression product thereof. For example, mutations may be introduced using techniques which are well known in the art, e.g., site-directed mutagenesis to insert new restriction sites, to alter glycosylation patterns or to change codon preference. By way of further example, the NOI may also be modified to optimise expression in a particular host cell. Other sequence changes may be desired in order to introduce restriction enzyme recognition sites.
  • the NOI may include within it synthetic or modified nucleotides.
  • a number of different types of modification to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule.
  • the NOI may be modified by any method available in the art. Such modifications may be carried out in to enhance the in vivo activity or life span of the NOI.
  • the NOI may be modified to increase intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences of the 5' and/or 3' ends of the molecule or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the backbone of the molecule.
  • One aspect of the present invention relates to amino acids that are immunologically reactive with the amino acid of SEQ ID No. 1.
  • Antibodies may be produced by standard techniques, such as by immunisation with the substance of the invention or by using a phage display library.
  • the term "antibody”, unless specified to the contrary, includes but is not limited to, polyclonal, monoclonal, chimeric, single chain, Fab fragments, fragments produced by a Fab expression library, as well as mimetics thereof.
  • Such fragments include fragments of whole antibodies which retain their binding activity for a target substance, Fv, F(ab') and F(ab') 2 fragments, as well as single chain antibodies (scFv), fusion proteins and other synthetic proteins which comprise the antigen-binding site of the antibody.
  • the antibodies and fragments thereof may be humanised antibodies. Neutralising antibodies, i.e., those which inhibit biological activity of the substance polypeptides, are especially preferred for diagnostics and therapeutics.
  • a selected mammal e.g., mouse, rabbit, goat, horse, etc.
  • the sequence of the present invention or a sequence comprising an immunological epitope thereof.
  • various adjuvants may be used to increase immunological response.
  • adjuvants include, but are not limited to, Freund's, mineral gels such as aluminium hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin,. and dinitrophenol.
  • BCG Bacilli Calmette- Gueri i
  • Corynebacterium parvum are potentially useful human adjuvants which may be employed if purified the substance polypeptide is administered to immunologically compromised individuals for the purpose of stimulating systemic defence.
  • Serum from the immunised animal is collected and treated according to known procedures. If serum containing polyclonal antibodies to the sequence of the present invention (or a sequence comprising an immunological epitope thereof) contains antibodies to other antigens, the polyclonal antibodies can be purified by immunoaffinity chromatography. Techniques for producing and processing polyclonal antisera are known in the art. In order that such antibodies may be made, the invention also provides polypeptides of the invention or fragments thereof haptenised to another polypeptide for use as immunogens in animals or humans.
  • Monoclonal antibodies directed against the sequence of the present invention can also be readily produced by one skilled in the art.
  • the general methodology for making monoclonal antibodies by hybridomas is well known.
  • Immortal antibody-producing cell lines can be created by cell fusion, and also by other techniques such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus.
  • Panels of monoclonal antibodies produced against orbit epitopes can be screened for various properties; i.e., for isotype and epitope affinity.
  • Monoclonal antibodies to the sequence of the present invention may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique originally described by Koehler and Milstein (1975 Nature 256:495-497), the human B-cell hybridoma technique (Kosbor et al (1983) Immunol Today 4:72; Cote et al (1983) Proc
  • Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening recombinant immunoglobulin libraries or panels of highly specific binding reagents as disclosed in Orlandi et al (1989, Proc Natl Acad Sci 86: 3833-3837), and Winter G and Milstein C (1991; Nature 349:293-299).
  • Antibody fragments which contain specific binding sites for the substance may also be generated.
  • fragments include, but are not limited to, the F(ab') 2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab') 2 fragments.
  • Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse WD et al (1989) Science 256:1275-128 1).
  • the amino acid sequence is used for large scale applications.
  • the amino acid sequence is produced in a quantity of from lg per litre to about 2g per litre of the total cell culture volume after cultivation of the host organism.
  • the amino acid sequence is produced in a quantity of from lOOmg per litre to about 900mg per litre of the total cell culture volume after cultivation of the host organism.
  • amino acid sequence is produced in a quantity of from 250mg per litre to about 500mg per litre of the total cell culture volume after cultivation of the host organism.
  • Figure 1 shows the separation of AFDH, APS1 and APS2 by hydrophobic interaction chromatography on a HiLoad Phenyl Sepharose 16/10 HP column (Pharmacia).
  • the solid line is absorbance at 280 nm (Y-axis right), the broken line is % of Buffer B (Y- axis left).
  • the elution volume is indicated in the X-axis.
  • the activity peaks are shaded.
  • the first activity peak is AFDH
  • the second is APS1
  • the third is APS2.
  • Figure 2 shows further purification of APS 1 by ion exchange chromatography on a 6 ml Resource column.
  • the solid line is absorbance at 280 nm (Y-axis right), the broken line is % of Buffer BI (Y-axis left).
  • the elution volume is indicated in the X-axis.
  • the three APS1 activity peaks are indicated (shaded areas).
  • Figure 3 shows polishing of APS 1 by gel filtration chromatography on Superdex 200. Absorbance at 280 nm was monitored (Y-axis). X-axis is the elution volume. APS1 activity was found with a elution volume of 15 ml in tube 15 to 17 which were pooled and concentrated.
  • Figure 4 shows electrophoresis of Ascopyrone P synthase (APS1). SDS-PAGE electrophoresis of the enzyme preparation after ion exchanger chromatography (lane 1, 2, 3, 5 and 6 from left), and the mol wt markers in kDa (lane 4). The gel was stained with PhastGel Blue R.
  • Figure 5 shows the purity examination of the purified APSl (lane 1 and 2 from left) and APS2 (lane 4)obtained by the gel filtration purification step. The analysis was
  • the Protein markers in kDa from Novex are from above: 116.3 (beta-galactosidase; 97.4(phosphorylase b); 66.3(BSA); 55.4(glutamic dehydrogenase); 36.5(lactate dehydrogenase); 31.0 (carbonic anhydrase).
  • Figure 6 shows the effect of pH on the activity of ascopyrone P Synthase 1 (APSl).
  • the buffers used were 0.5 ml of sodium acetate (pH 4.0 to 5.4), sodium phosphate (pH 5.7 to 8.0), and Tris-HCl (pH 8.4 to 9.0).
  • the reaction mixture had a total volume 0.7 ml and the reaction time was 40 min, reaction temperature was 22 °C. Other factors were th ⁇ same as in the methodlsection below.
  • Figure 7 shows the effect of temperature on the activity of ascopyrone P Synthase 1 (APSl) using ascopyrone M (APM) as substrates in 50m m sodium acetate (pH 5.4).
  • Figure 8 shows the effect of substrate concentration on the APSl activity.
  • Ascopyrone P synthase 1 was purified by a simple and efficient purification procedure from A. melaloma. A purification of 408 fold was achieved. APSl was apparently a homodimer as a molecular mass of 60 kDa was observed in SDS-gel electrophoresis using gels with 8-25% gradient and 124 kDa on gel filtration chromatography by a Superdex-200 column. The purified APSl had a specificity of 3878 ⁇ mol ascopyrone P min "1 mg "1 protein. The concentration of the substrate ascopyrone M (APM) that yielded half of the maximum activity was 0.405 ⁇ M, Nmax was estimated to be 4.494 units.
  • APM substrate ascopyrone M
  • APSl had an optimal pH-range of 5.0 to pH 6.0 with the optimal activity at pH 5.5. APSl had a wide temperature optimum range from 25°C to 50°C with an optimum temperature at 48°C.
  • Several isoforms of ascopyrone P synthase were present in the cell-free extract. Ascopyrone P synthase was resolved in two isoforms (APSl and APS2) in the hydrophobic interaction chromatography step and additionally APSl into 3 isoforms in the ion-exchange chromatography step. APS2 was purified and showed the same molecular mass of 60kDa as APSl on SDS-PAGE.
  • APS2 was found to be AINLPFSNWAX(or G)TI by amino acid sequencing of the purified APS2.
  • APSl was found to contain the sequence
  • the reaction mixture consisted of- 50 ⁇ l AF (30 mg ml "1 ), 10 to 50 ⁇ l AFDH sample, 0.5 ml 50 mM sodium phosphate buffer (pH 7.5) containing 1.0 M ⁇ aCl and deionized water to a total volume of 0.7 ml.
  • the reaction mixture was vortexed and incubated at 22 °C for 30 min.
  • the reaction mixture was scanned between 400-200 nm and the peak absorbance at 263nm was recorded on a Perkin Elmer Lambda 18 uv/vis spectrophotometer.
  • One activity unit of AFDH is defined an increase of 0.01 absorbance unit at 263 nm at 22 °C per min.
  • AFDH product was prepared in the same way as for the activity assay of AFDH except that more AF (final AF concentration 2-4%) was used and the reaction was performed in a membrane-reactor with a molecule cutoff of 10,000. The reaction was followed by the increase at 263 nm. At the end of reaction the AFDH product formed was separated form the AFDH and used for the assay of APS.
  • the coupled reaction assay method was used with AFDH as tool enzyme:
  • the reaction mixture consisted of 50 ⁇ l AF (30 mg/ml), l ⁇ l of AFDH, 0.5 ml 50 mM ⁇ a-Phosphate buffer (pH 7.5) containing 1.0 M ⁇ aCl, and 149 ⁇ l deionised water to a total volume of 0.7 ml.
  • the reaction mixture was vortexed and incubated at 22°C for 30 minutes to convert AF to ascopyrone (APM).
  • APM ascopyrone
  • the reaction mixture was passed through a centriprep-10 filter with a molecular cut-off of 10000 to separate the enzyme from the APM formed.
  • 10 ⁇ l sample of APS was added, mixed and incubated at 22°C for 30 minutes.
  • the reaction mixture was scanned between 400-200 nm and the peak absorbance at 289 nm was recorded on a
  • Perkin Elmer lambda 18 uv/vis spectrophotometer One activity unit of APS is defined as the enzyme needed to produce 1 ⁇ mol APP at 22°C per minute.
  • the direct assay method the same as the coupled reaction assay method except AF and AFDH were replaced with the product of AFDH prepared from the enzyme reactor.
  • One activity unit of APS is defined an increase of 0.01 absorbance unit at 289 nm at 22 °C per min.
  • the assay methods for ADH and APS were also adapted to use a microplate and microplate reader.
  • the reaction volume for AFDH and APS was reduced to 0.2 ml.
  • 10 ⁇ l 1 N NaOH was added to each well of the microplate to stop the reaction and APP content was measured at 340 nm using a microplate reader (Model EAR 340 AT, SLT-Labinstruments, Gr ⁇ dig, Austria).
  • the reaction mixture contained also APS as a tool enzyme. This method is used for fast screening the activities of AFDH and APS, such identifying the activity fractions in the chromatography steps.
  • the fungus A. melaloma (CBS 293.54) was obtained from Centraalbureau voor Schimmelcultures (CBS, Baarn, NL).
  • A. melaloma was grown on PDA medium for 20 days at 24°C. To induce AFDH and APS production the mycelium was carefully removed from the agar plates and placed at -20°C for 24 hours. The biomass of 854 g was thawed at room temperature (22-24°C). 500 ml of 50 mM Na-phosphate (pH 7.5) and 1 % of toluene was added to the biomass, mixed and placed at 22°C for 3 hours and then homogenized with an ultraturax for at least 15 minutes.
  • the mixture was then incubated at 4°C.fo 24 hours.
  • the mixture was then centrifuged at ' ⁇ 0000 x g at 4°C for 30 minutes and the supernatant was filtered through a whatman filter paper. A total volume of 500 ml was obtained.
  • Ammonium sulphate was added slowly to the supernatant to 40% saturation at 0°C and after 30 minutes at 0°C, the solution was centrifuged at 10000 x g for 30 minutes. To the supernatant ammonium sulphate was added to 80% saturation.- After 30 minutes at 0°C, the solution was centrifuged at 10000 rpm for 30 minutes. The supernatant was carefully removed and the pellet was resuspended in 54 ml 50 mM Na-phosphate buffer (PH 7.5).
  • the column was washed with buffer Al and eluted with a stepwise gradient (linear gradient from 0- 55 % buffer BI in 10 column volumes, followed by 55% buffer Bl for 5 column volumes, and then from 55 % to 100 % Buffer Bl linearly in 10 column volumes.
  • the column was cleaned with 100%) buffer Bl in 3 column volumes (Fig 1).
  • the flow rate was 2 ml/min. Fraction size 3 ml.
  • Active fractions of APSl was pooled (55 ml) and concentrated with centriprep 10 with a molecule cutoff of 10000 (Millipore incorporation, USA).
  • the sample was desalted on a PD-10 gel filtration columns (Pharmacia) and the buffer was changed to 20 mM Bis-Tris-Propane -HCl buffer (pH 7.5) (buffer A2).
  • This step is a polishing step and for measuring of the molecular mass of APSl.
  • the pooled and concentrated active APSl fractions (15-17) were loaded onto a gel filtration column of Superdex 200 column (Pharmacia). The column was pre-equilibrated and eluted with 50 mM Na-Phosphate buffer (pH 7.0) containing 0.1 M NaCl (Fig 3). APSl Peak, fraction 15-17, was pooled and concentrated.
  • the column was calibrated using Pharmacia's gel filtration protein markers of ribonuclease A (13700), ovalbumin (43000), albumin (67000) and aldolase (158000).
  • the void volume was determined using bluedextran.
  • the molecular mass of APSl was estimated to be 124 kDa from its distribution coefficient, relative to the marker proteins. A summary of the purification is given in Table 1. 4. Characterization of APSl
  • APS purification of APS procedure was followed by SDS-Page, and native-page using Phastsystem (Pharmacia) and pre-cast gels with a gel gradient of 8-25% according to the manufacturer's instructions. Nisualization of protein bands on the gels was made with silvers staining (silverxpress, Invitrogen) (Fig 4). The mol mass of APSl and APS2 were determined by SDS-PAGE (Fig. 4) to be both 60kDa.
  • Table 1 A summary of the purification steps for APSl from Anthracobia melaloma.
  • APS was further purified and efficiently separated from AFDH by hydrophobic interaction chromatography on HiLoad Phenyl Sepharose 16/10 HP (Fig. 1).
  • APS was resolved into two isoforms (APSl and APS2).
  • AFDH was first eluted with 39 % Buffer B, followed by APP Synthase 1 (APS 1) at 55% Buffer B, APP Synthase 2 (APS2) at 100% Buffer B. It is remains unknown whether APSl and APS2 are coded by the same gene or by different genes.
  • the activity ratio of APSl to APS2 was around 2:3.
  • APSl was further resolved into 3 active peaks eluted at respectively 5%, 10% and 12% Buffer Bl (Fig. 2).
  • the first peak had 72%, second peak 23% and third peak 5% of the total APSl activity.
  • As the first peak was the major APSl peak, it has been used for further characterization (amino acid sequencing, optimum- pH, temperature Km,-salt effect etc).
  • the first fraction of APSl from the ion exchanger step was analysed on a gel filtration column Superdex 200 (Fig. 3). APSl was found in the first major peak with an elution volume of 15 ml. The second peak was a non-proteinaceous substances.
  • the column was calibrated using Pharmacia's gel filtration protein markers of ribonuclease A (13,700), ovalbumin (43,000), albumin (67,000), aldolase (158,000), catalase (232,000), ferritin (440,000), and thyroglobulin (669,000).
  • the void volume was determined using blue dextrin. A molecular mass of 158 kDa was estimated for APSl from its partition coefficient relative to the marker proteins.
  • the first fraction of APSl from the ion exchange step and gel filtration step showed a molecular mass of 60 kDa on 8-25% gradient gel of SDS-PAGE. The same value was obtained for APSl from the gel filtration step (Fig. 4). Furthermore all the three fractions of APSl resolved on the ion-exchange step showed one single band with a relative molecular mass (Mr) of 53 kDa. APS2 showed also this molecular mass of around 60 kDa. 5.2. Characterization of APS
  • APSl activity increased with the. increase of the concentration of salts.
  • APSl activity increased in the presence of NaCl at concentration at least up to 0.5 M.
  • the reaction mixture was vortexed and incubated at 22°C for 15 min.
  • the activity of APSl was measured as described earlier (Table 2).
  • the optimum temperature and stability of APP synthasel The. reaction mixture consisted of 500 ⁇ L 50 mM Hac-NaAc (pH 5.0), 100 ⁇ L APM substrate (2.46 mg/ml), deionized water to a total volume of 0.7 ml and 1 ⁇ L of APSl (271.13 units/ml). The reaction mixtures were vortexed and incubated 15 minutes at different temperatures (4°C-60°C). APSl activity was measured as described earlier (Table 3). Table 3. The effect of the temperature on the activity of APSl.
  • the cell-free extract of the A. melaloma did not lose its APS activity for 20 days at 4°C.
  • the activity of APSl was measured as a function of the APM concentration.
  • the reaction mixtures for APSl consisted of 500 ⁇ l 50 mM Na-Phosphate buffer (pH 7.5) containing 1.0 M NaCl, 5-400 ⁇ l substrate APM (6.124 ⁇ mol/ml), deionized water to a total volume of 1.4 ml and l ⁇ l of APSl (271,13 units/ml) was added to the mixture. The reaction mixture was vortexed and incubated at 22°C for 30 min (Table 4).
  • APS was further purified and efficiently separated from AFDH by hydrophobic interaction chromatography on HiLoad Phenyl Sepharose 16/10 HP (Fig 1). Furthermore APS was resolved into two isoforms (APSl and APS2). AFDH was first eluted with 39% buffer Bl, followed by APP synthase 1 (APSl) at 55 % buffer Bl, APP synthase 2 (APS2) at 100 % buffer Bl. The activity ratio of APSl to APS2 was around 2:3 (Fig. 1).
  • APSl was further resolved into 3 active peaks eluted at respectively 5 %, 10 % and 12 % buffer B2 (Fig 2).
  • the first peak had 72 %, second peak 23 % and the third peak 5 % of the total APSl activity.
  • As the first peak was the major APS 1 peak, it was used for further characterization. 6.1.4 Gel filtration chromatography
  • the first fraction of APSl from the ion exchange step was concentrated and analyzed on a gel filtration column Superdex 200 (Fig 10) APSl was found in the first major peak with an elution volume of 15 ml.
  • the column was calibrated using Pharmacia's gel filtration protein markers.
  • the void volume was determined by blue dextrin to be 9 ml (Table 7).
  • a molecular mass of 124 kDa was estimated for APSl from its partition coefficient relative to the marker proteins.
  • APS2 ws found to be AI ⁇ LPFS ⁇ WAX (or OTI by amino acid sequencing of the purified APS2.
  • APS 1 was found to contain the sequence EYGRTFFTRYDYE ⁇ ND.
  • the APSl from Anthracobia melaloma had an optimal pH range of 5.0 to pH 6.0 with the optimal activity at pH 5.5 (Table 2).
  • the enzyme activity decreased dramatically in pH values lower than 4.5 and higher than 6.3.
  • APP synthase 1 showed similar activity in Mes-NaOH (5.5-6.7), Mops-NaOH (6.0-8.0) and Bicine-NaOH (7.6-9.0).
  • APP synthasel had a wide temperature optimum range, from 25°C to 50°C with an optimum temperature at 48°C when a reaction time of 15 min was used. At temperatures above i ⁇ 3G " °G the activity of APP synthase decreased rapidly (Table ⁇ ).
  • the purified enzyme was made in 50 mM Na-Phosphate buffer (pH 7.0) 0.1 M NaCl, no activity loss was observed for 30 days at 4°C.
  • the cell-free extract did not lose its APS and AFDH activity when stored at 4°C for 20 days.
  • Antibodies were raised against the amino acid of the present invention by injecting rabbits with the purified enzyme and isolating the immunoglobulins from antiserum according to procedures described according to N Harboe and A Ingild ("Immunization, Isolation of Immunoglobulins, Estimation of Antibody Titre" In A Manual of Quantitative Immunoelectrophoresis, Methods and Applications, N H Axelsen, et al (eds.), Universitetsforlaget, Oslo, 1973) and by T G Cooper ("The Tools of Biochemistry", John Wiley & Sons, New York, 1977).

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Abstract

La présente invention concerne la purification et la caractérisation de l'ascopyrone P synthase.
PCT/GB2002/004885 2001-10-31 2002-10-30 Ascopyrone p synthase WO2003038084A1 (fr)

Applications Claiming Priority (4)

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GBGB0126163.5A GB0126163D0 (en) 2001-10-31 2001-10-31 Sequences
GB0126163.5 2001-10-31
US34331301P 2001-12-21 2001-12-21
US60/343,313 2001-12-21

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004039993A1 (fr) * 2002-10-30 2004-05-13 Danisco A/S Polynucleotide codant une pyranosmome deshydratase
WO2003038107A3 (fr) * 2001-10-31 2004-10-28 Danisco Procede
WO2005040147A1 (fr) 2003-10-28 2005-05-06 Nihon Starch Co., Ltd. Antitumoral
US7517981B2 (en) 2003-11-20 2009-04-14 Nihon Starch Co., Ltd. Efficient production method of ascopyrone P

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BAUTE M-A ET AL: "ENZYME ACTIVITY DEGRADING 1,4-ALPHA-D-GLUCANS TO ASCOPYRONES P AND T IN PEZIZALES AND TUBERALES", PHYTOCHEMISTRY, PERGAMON PRESS, GB, vol. 33, no. 1, 1993, pages 41 - 45, XP000925242, ISSN: 0031-9422 *
THOMAS L V ET AL: "Ascopyrone P, a novel antibacterial derived from fungi.", JOURNAL OF APPLIED MICROBIOLOGY, vol. 93, no. 4, 2002, 2002, pages 697 - 705, XP002235137, ISSN: 1364-5072 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003038107A3 (fr) * 2001-10-31 2004-10-28 Danisco Procede
WO2004039993A1 (fr) * 2002-10-30 2004-05-13 Danisco A/S Polynucleotide codant une pyranosmome deshydratase
WO2005040147A1 (fr) 2003-10-28 2005-05-06 Nihon Starch Co., Ltd. Antitumoral
US7649016B2 (en) 2003-10-28 2010-01-19 Nihon Starch Co., Ltd. Antitumor medicine
US7517981B2 (en) 2003-11-20 2009-04-14 Nihon Starch Co., Ltd. Efficient production method of ascopyrone P
CN100480246C (zh) * 2003-11-20 2009-04-22 日本淀粉工业株式会社 子囊吡喃酮p的制造方法

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