[go: up one dir, main page]

WO1997033984A1 - Nouveaux variants de protease d'achromobacter lyticus - Google Patents

Nouveaux variants de protease d'achromobacter lyticus Download PDF

Info

Publication number
WO1997033984A1
WO1997033984A1 PCT/DK1997/000100 DK9700100W WO9733984A1 WO 1997033984 A1 WO1997033984 A1 WO 1997033984A1 DK 9700100 W DK9700100 W DK 9700100W WO 9733984 A1 WO9733984 A1 WO 9733984A1
Authority
WO
WIPO (PCT)
Prior art keywords
replaced
protease
amino acid
lysine
alkyl
Prior art date
Application number
PCT/DK1997/000100
Other languages
English (en)
Inventor
Asser Andersen
Per Balschmidt
Sven Branner
Sven Hastrup
Original Assignee
Novo Nordisk 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
Application filed by Novo Nordisk A/S filed Critical Novo Nordisk A/S
Priority to AU20913/97A priority Critical patent/AU2091397A/en
Publication of WO1997033984A1 publication Critical patent/WO1997033984A1/fr

Links

Classifications

    • 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
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea

Definitions

  • the present invention relates to novel polypeptides with Achromobacter lyticus protease (API) activity, nucleic acid constructs encoding the polypeptides, and recombinant vectors and recombinant host cells comprising the nucleic acid constructs.
  • API Achromobacter lyticus protease
  • proteolytic enzymes proteolytic enzymes
  • Pepsin the important digestive enzymes
  • Trypsin the important digestive enzymes
  • Chymotrypsin the important digestive enzymes
  • proteases of animal, plant and microbial origin are known and several have found sufficient application to be produced industrially. These applications include applications for medical purposes, for food processing and laundering purposes as well as applications in protein synthesis and structural analysis.
  • proteases proteases are characterized by specificity, high yield and stability of the product (peptide fragments and amino acids) to the reagent (protease).
  • Lysyl Endopeptidase is a protease secreted by the soil bacterium Achromobacter lyticus (cf. Masaki et al., Agric.Biol.Chem. 42, 1978, p. 1443).
  • the protease is identical to the enzyme discovered by Masaki et al., and named Achromobacter protease I (cf. Masaki et al., Biochem.Biophvs.Acta 660, 1981, p. 44).
  • Japanese patent application No.64-02574 (Wako) (priority from 870622) relates to a method for purification of Achromobacter protease I.
  • the primary structure of Achromobacter lyticus protease I appears from S.
  • API is a trypsin-like serine protease which specifically cleaves the peptide bonds (-Lys-X-) at the side of the carboxyl groups of lysine residues in proteins and peptides, and is also called a lysyl endopeptidase (EC 3.4.21.50).
  • This enzyme cleaves all Lys-X bonds including the Lys-Pro bond, and therefore has been used for the fragmentation of proteins or peptides for their primary structural analysis, the preparation of peptide maps, and the synthesis of Lys-X-compounds.
  • API is presently used for cleaving fusion proteins or producing des-B30-insulin.
  • European patent No. 17.938 (Shionogi) (priority from 790413) relates to a method for producing B30-Thr-insulin by reacting a derivative of threonine and des-B30-insulin with a protease, e.g. A.lyticus protease I.
  • a protease e.g. A.lyticus protease I.
  • 57-67548 (Shionogi) (priority from 801014) relates to a method for producing insulin analogues in which the B30 amino acid residue has been substituted with another amino acid residue by using trypsin, a trypsin-like protease or A.lyticus protease I.
  • European patent No. 92.829 (Wako) (priority from 820423) relates to the preparation of human insulin derivatives from porcine insulin by using A.lyticus protease I or a water-soluble cross-linked A.lyticus protease I. European patent application No.
  • 206.769 and 440.311 (Fujisawa) (priority from 850620) relates to the use of Achromobacter protease I for cleaving fusion proteins having a lysine between a protective peptide and a target peptide (e.g. human atrial natriuretic polypeptide (hANP)) containing no lysine residues.
  • European patent No. 354.507 (Hoechst) (priority from 880810) relates to a method for producing des-B30-insulin by using a protease, e.g. A.lyticus protease I.
  • European patent application No. 387.646 (Wako) (priority from 890314) relates to a novel DNA encoding Achromobacter protease I for recombinant production of enzyme, and for fragmentation of protein(s) and peptide, for peptide mapping and synthesis of Lys-X- compounds.
  • WO 96/17943 (Novo Nordisk A/S) (priority from 941209) relates to a method of producing an extracellular protein, e.g. API, in a bacterium transformed with a DNA sequence encoding the API.
  • One object of this invention is to make available an enzyme which specifically splits a protein or peptide at the C-terminal end of lysine and which has a higher activity than Achromobacter lyticus protease I.
  • a further object of this invention is to make available an enzyme which specifically splits a protein or peptide at the C-terminal end of lysine and which has a higher stability than Achromobacter lyticus protease I.
  • a further object of this invention is to make available an enzyme which specifically splits a protein or peptide at the C-terminal end of lysine and which has a better performance than Achromobacter lyticus protease I when immobilized on a water-insoluble earner.
  • the term "Achromobacter lyticus protease I variant” designates an enzyme which specifically splits a protein or peptide at the C-terminal end of lysine and which has a homology with Achromobacter lyticus protease I of more than 90%, preferably more than 95%. Accordingly, the present invention relates to novel Achromobacter lyticus protease I variants wherein one or more of the lysine residues in positions 30, 49, 106, 155 and 203 (referring to Fig. No.1) have been replaced by another amino acid residue which can be encoded by nucleic acid constructs, or wherein one or more of the lysine residues in the above mentioned positions or other amino acid residues introduced into the above mentioned positions have been chemically modified.
  • the present invention relates to a A.lyticus protease I variant immobilized on a water-Insoluble carrier with or without the use of a spacer or linker molecule.
  • the present invention relates to a cross-linked polymer of a A.lyticus protease I variant, or a protease variant cross-linked to a carrier protein with or without the use of a spacer or linker molecule.
  • the present invention relates to nucleic acid constructs comprising a nucleotide sequence, preferably a DNA sequence, encoding said Achromobacter lyticus protease variants.
  • the present invention relates to recombinant vectors comprising the said nucleic acid constructs.
  • the present invention relates to recombinant host cells comprising said nucleic acid constructs or said vectors.
  • Fig.1 shows the amino acid sequence of unmodified API.
  • Fig.2 shows the gene structure of the unmodified A.lyticus protease I gene.
  • Fig.3 A-C show the general amino acid sequence of the novel API-derivates according to the invention, where Xaa designates an amino acid residue which may be encoded by a nucleic acid construct, or Xaa designates a lysine residue which has been chemically modified, and, in particular, [K30RJ-API (SEQ ID NO:1) and [Me r K30]-API (SEQ ID NO:2).
  • Fig.4 A-B show plasmid charts of A.lyticus protease I expression plasmid, pSX547, and A.lyticus protease I K30R expression plasmid, pSX582.
  • Fig.5 shows the processing of preproinsulin into des-B30-insulin using methylated API ([Me K30J-API).
  • Fig.6 shows the processing of preproinsulin into des-B30-insulin using [K30RJ-API.
  • Fig.7 A-B shows plasmid charts illustrating the preparation of A.lyticus protease I expression plasmid pSX547.
  • the gene coding for A.lyticus protease I encodes a polypeptide of 653 amino acid residues. This includes a signal (or pre) peptide of 20 amino acids, an N-terminal propeptide of 185 amino acids, a core (or mature) protein of 268 amino acids which is the active protease, and a C-terminal propeptide of 180 amino acids, as shown in figs. 1 and 2 (cf. T.Ohara et al., J.Biol.Chem. 264 (34), 1989, pp. 20625-20631).
  • API active protease
  • the present invention relates to novel Achromobacter lyticus protease I variants wherein one or more of the lysine residues in positions 30, 49, 106, 155 and 203 (referring to Fig. 1) have been replaced by another amino acid residue which can be encoded by nucleic acid constructs, or wherein one or more of the lysine residues in the above mentioned positions or other amino acid residues introduced into the above mentioned positions have been chemically modified.
  • the present invention relates to Achromobacter lyticus protease I denvatives compnsing the ammo acid sequence shown i Fig 1 wherein one or more of the lysine residues in positions 30, 49, 106, 155 and 203 are - replaced by an ammo acid residue different from lysine which is positively charged at neutral pH,
  • R is hydrogen or-C(1-6)-alkyl
  • R 1 is -C(1-6)-alkyl, or-CNH-NR'R", or-CNH-R', where R' and R" are different or identical, and are H or C(1-6)-alkyl
  • N-terminal ammo group may be modified as well
  • C(1-6)-alkyl refers to a straight or branched, saturated hydrocarbon chain having 1 to 6 carbon atoms, such as e g methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, 2-methylbutyl, 3- methylbutyl, n-hexyl, 4-methylpentyl, neopentyl, and 2,2-d ⁇ methylpropyl
  • one or more of the lysine residues are replaced by a hydrophilic ammo acid which is non-charged at neutral pH, e g Gly, Asn, Gin, Ser, or Thr, or replaced by a lysine residue with the formulae [-NH-CH[(CH 2 ) 4 -NRR 1 ]-CO-], in which R is hydrogen or C(1-6)-alkyl, and R 1 is -CO-NR'R", where R' and R" are different or identical, and are hydrogen or C(1-6)-alkyl, or R 1 is -CO-R 2 , where R 2 is C(1-6)-alkyl.
  • one or more of the lysine residues in the API protease are replaced by a hydrophobic amino acid which is uncharged at neutral pH, e.g. He, Leu, Val, Ala, Phe, Tyr, Tip, Pro, Cys, or Met.
  • a hydrophobic amino acid which is uncharged at neutral pH, e.g. He, Leu, Val, Ala, Phe, Tyr, Tip, Pro, Cys, or Met.
  • one or more of the lysine residues in the API protease are replaced by an amino acid different from lysine which is positively charged at neutral pH, e.g. Arg, or His, or replaced by a modified lysine residue with the general formula [-NH- CH[(CH 2 ) 4 -NRR 1 ]-CO-], in which R is hydrogen or C(1-6)-alkyl, and R is C(1-6)-alkyl, or -CNH- NR'R", or -CNH-R', where R' and R" are different or identical, and are hydrogen or C(1-6)- alkyl.
  • one or more of the lysine residues in the API protease are replaced by an arginine residue or a residue with the general formula [-NH-[(CH 2 ) 4 -NMe 2 ]-CO-].
  • lysine residue in position 30 is modified.
  • lysine residue in position 49 is modified.
  • the lysine residue in position 106 is modified.
  • the lysine residue in position 155 is modified.
  • the lysine residue in position 203 is modified.
  • protease is [K30RJ-API (SEQ ID NO:1) or [Me 2 -K30]-API (SEQ ID NO:2).
  • the amino acid in position 30 in API is probably the most readily accessible lysine residue from an sterical and electrostatically point of view.
  • the distance from this lysine residue to the active site of the enzyme is rather short (approximately 0.9 nm). This indicates that this amino acid in particular is very important for the accessibility of substrate to the active site of the enzyme, and that the amino acid in position 30 in particular has influence on the specific activity of the enzyme.
  • the Achromobacter lyticus protease variants according to the present invention are useful for cleavage of polypeptides.
  • Polypeptides suitable for cleavage may be fused polypeptides, or polypeptides such as preproinsulins or analogues hereof, preferably preproinsulins.
  • the preproinsulins may be of human, porcine, or bovine origin, preferably human preproinsulin.
  • the modified protease may be used in solution; alternatively, the protease may be cross- linked with conventional cross-linking agents (for instance glutaraldehyde, diisocyanate, formaldehyde, etc.) to convert it into a polymer; or the protease may be cross-linked to a carrier polypeptide.
  • the protease may be immobilised on a soluble matrix, such as a polyglutamic acid matrix, by conventional procedures being well known to persons skilled in the art.
  • the modified API may be immobilized on a solid phase when used for cleavage of polypeptides.
  • the solid phase may be a cellulose matrix, a polysaccharide matrix such as a cellulose or cross-linked agarose matrix, a silica matrix, a dextran matrix, a polyacrylamide matrix, a polyamide matrix, (e.g. sephadex or sepharose), preferably a polysaccharide matrix.
  • preactivated polysaccharide matrixes include Mini-LeakTM and CNBr- SepharoseTM
  • preactivated polyacrylamide matrixes include EupergitTM and Affi-
  • the enzyme When immobilized or cross-linked, the enzyme may be attached to the matrix or carrier protein via a spacer or linker.
  • a spacer or linker may be a polyfunctional organic compound. Suitable spacers or linkers are well known to persons skilled in the art, as are the methods for employing such spacers or linkers.
  • the protease according to the present invention may be produced by post-translational chemical modification of the "natural" protease, or by site directed mutagenesis of the A.lyticus protease encoding nucleic construct.
  • Lysine is an amino acid having a second amino entity located in ⁇ -position.
  • Such amino groups are basic groups and are positively charged except at high pH.
  • uncharged form amino groups are powerful nucleophiles and are capable of being chemically modified in several ways, such well known modifications being, e. g., reaction with carboxylic acid anhydride (e.g. acetic acid anhydride, succinic acid anhydride, maleic acid anhydride), reaction with cyanate (carbamylation) giving substituted ureas, reaction with isoureas (guadination), reaction with imidates (aminidation), and reductive alkylation.
  • carboxylic acid anhydride e.g. acetic acid anhydride, succinic acid anhydride, maleic acid anhydride
  • reaction with cyanate carboxylic acid anhydride
  • carboxylic acid anhydride e.g. acetic acid anhydride, succinic acid anhydride, maleic acid anhydride
  • nucleic acid construct is intended to indicate any nucleic acid molecule of cDNA, genomic DNA, synthetic DNA or RNA origin.
  • construct is intended to indicate a nucleic acid segment which may be single- or double-stranded, and which may be based on a complete or partial naturally occurring nucleotide sequence encoding a polypeptide of interest.
  • the construct may optionally contain other nucleic acid segments.
  • the nucleic acid construct of the invention encoding the polypeptides of the invention may suitably be of genomic or cDNA origin, for instance obtained by preparing a genomic or cDNA library and screening for DNA sequences coding for all or part of the polypeptide by hybridization using synthetic oligonucleotide probes in accordance with standard techniques (cf. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd. Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989).
  • the nucleic acid construct of the invention encoding the polypeptide may also be prepared synthetically by established standard methods, e.g. the phosphoamidite method described by Beaucage and Caruthers, Tetrahedron Letters 22 (1981), 1859 - 1869, or the method described by Matthes et al., EMBO Journal 3 (1984), 801 - 805.
  • phospho ⁇ amidite method oligonucleotides are synthesized, e.g. in an automatic DNA synthesizer, purified, annealed, ligated and cloned in suitable vectors.
  • nucleic acid construct may be of mixed synthetic and genomic, mixed synthetic and cDNA or mixed genomic and cDNA origin prepared by ligating fragments of synthetic, genomic or cDNA origin (as appropriate), the fragments corresponding to various parts of the entire nucleic acid construct, in accordance with standard techniques.
  • the nucleic acid construct may also be prepared by polymerase chain reaction (PCR) using specific primers, for instance as described by Sambrook et al., supra, or as described in US 4,683,202 or Saiki et al., Science 239 (1988), 487 - 491.
  • PCR polymerase chain reaction
  • the DNA sequence encoding the prepropeptide may be prepared by PCR amplification of chromosomal DNA of the species from which the the prepropeptide is derived.
  • the DNA sequence encoding the desired protein may be prepared by PCR amplification of chromosomal DNA of the species from which the protein is derived, or for instance by screening a genomic or cDNA library with oligonucleotides as indicated above.
  • the nucleic acid construct of the invention comprises nucleic acid sequences encoding the amino acid sequence shown in SEQ ID NO:1 and SEQ ID NO:2.
  • the nucleic acid construct is preferably a DNA construct which term will be used exclusively in the following.
  • the present invention relates to a recombinant vector comprising a DNA construct of the invention.
  • the recombinant vector into which the DNA construct of the invention is inserted may be any vector which may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced.
  • the vector may be an autonomously replicating vector, i.e. a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid.
  • the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.
  • the vector is preferably an expression vector in which the DNA sequence encoding the polypeptide of the invention is operably linked to additional segments required for transcription of the DNA.
  • the expression vector is derived from plasmid or viral DNA, or may contain elements of both.
  • operably linked indicates that the segments are arranged so that they function in concert for their intended purposes, e.g. transcription initiates in a promoter and proceeds through the DNA sequence coding for the polypeptide.
  • the expression vector is the plasmid pSX582 as described in Example 2 and Fig.4B.
  • the promoter may be any DNA sequence which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell.
  • suitable promoters for directing the transcription of the DNA encoding the polypeptide of the invention in mammalian cells are the SV40 promoter (Subramani et al., MoL Cell BjoL 1 (1981), 854 -864), the MT-1 (metallothionein gene) promoter (Palmiter et al., Science 222 (1983), 809 - 814) or the adenovirus 2 major late promoter.
  • promoters for use in yeast host cells include promoters from yeast glycolytic genes (Hitzeman et al., J BjoL Chem. 255 (1980), 12073 - 12080; Alber and Kawasaki, J, MoL Appl. Gen. 1 (1982), 419 - 434) or alcohol dehydrogenase genes (Young et al., in Genetic Engineering of Microorganisms for Chemicals (Hollaender et al, eds.), Plenum Press, New York, 1982), or the TPM (US 4,599,311) or ADH2-4c (Russell et al., Nature 304 (1983), 652 - 654) promoters.
  • suitable promoters for use in bacterial host cells include the promoter of the Bacillus stearothermophilus maltogenic amylase gene, the Bacillus licheniformis alpha-amylase gene, the Bacillus amyloliquefaciens BAN amylase gene, the Bacillus subtilis alkaline protease gen, or the Bacillus subtilis xylosidase gene, or by the phage Lambda P R or P L promoters or the E. coli lac, tijj or tac promoters.
  • the DNA sequence encoding the polypeptide of the invention may also, if necessary, be operably connected to a suitable terminator.
  • the vector may further comprise elements such as polyadenylation signals, transcriptional enhancer sequences, and translational enhancer sequences
  • the recombinant vector of the invention may further comprise a DNA sequence enabling the vector to replicate in the host cell in question.
  • the vector may also comprise a selectable marker, e.g. a gene the product of which complements a defect in the host cell, such as the gene coding for dihydrofolate reductase (DHFR) or the Schizosaccharomyces pombe TPI gene (described by P.R. Russell, Gene 40, 1985, pp. 125-130), or one which confers resistance to a drug, e.g. ampicillin, kanamycin, tetracyclin, chloramphenicol, neomycin, hygromycin or methotrexate.
  • DHFR dihydrofolate reductase
  • Schizosaccharomyces pombe TPI gene described by P.R. Russell, Gene 40, 1985, pp. 125-130
  • a drug e.g. ampicillin, kanamycin, tetra
  • a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) may be provided in the recombinant vector.
  • the secretory signal sequence is joined to the DNA sequence encoding the polypeptide in the correct reading frame.
  • Secretory signal sequences are commonly positioned 5' to the DNA sequence encoding the polypeptide.
  • the secretory signal sequence may be that normally associated with the polypeptide or may be from a gene encoding another secreted protein.
  • the secretory signal sequence may encode any signal peptide which ensures efficient direction of the expressed polypeptide into the secretory pathway of the cell.
  • the signal peptide may be naturally occurring signal peptide, or a functional part thereof, or it may be a synthetic peptide. Suitable signal peptides have been found to be the ⁇ -factor signal peptide, the signal peptide of mouse salivary amylase (cf. O. Hagenbuchle et al., Nature 289, 1981, pp. 643-646), a modified carboxypeptidase signal peptide (cf. L.A. Vails et al., Cell 48, 1987, pp. 887-897), the yeast BAR1 signal peptide (cf. WO 87/02670), or the yeast aspartic protease 3 (YAP3) signal peptide (cf. M. Egel-Mitani et al., Yeast 6, 1990, pp. 127-137).
  • a sequence encoding a leader peptide may also be inserted downstream of the signal sequence and upstream of the DNA sequence encoding the polypeptide.
  • the function of the leader peptide is to allow the expressed polypeptide to be directed from the endoplasmic reticulum to the Golgi apparatus and further to a secretory vesicle for secretion into the culture medium (i.e. exportation of the polypeptide across the cell wall or at least through the cellular membrane into the periplasmic space of the yeast cell).
  • the leader peptide may be the yeast ⁇ -factor leader (the use of which is described in e.g.
  • the leader peptide may be a synthetic leader peptide, which is to say a leader peptide not found in nature. Synthetic leader peptides may, for instance, be constructed as described in WO 89/02463, WO 92/11378, or WO 95/34666.
  • the DNA sequence encoding the present polypeptide introduced into the host cell may be either homologous or heterologous to the host in question. If homologous to the host cell, i.e. produced by the host cell in nature, it will typically be operably connected to another promoter sequence or, if applicable, another se ⁇ etory signal sequence and/or terminator sequence than in its natural environment.
  • the term "homologous” is intended to include a cDNA sequence encoding a polypeptide native to the host organism in question.
  • heterologous is intended to include a DNA sequence not expressed by the host cell in nature. Thus, the DNA sequence may be from another organism, or it may be a synthetic sequence.
  • the host cell into which the DNA construct or the recombinant vector of the invention is introduced may be any cell which is capable of producing the present polypeptide and includes bacteria, yeast, fungi and higher eukaryotic cells.
  • bacterial host cells which, on cultivation, are capable of producing the polypeptide of the invention are grampositive bacteria such as strains of Bacillus, such as strains of B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearother ophilus, B. al alophilus, B. amyloliquefaciens, B. coagulans, B. circulans, B. lautus, B. megatherium or B. thuringiensis, or strains of Streptomyces, such as S.
  • the transformation of the bacteria may be effected by protoplast transformation or by using competent cells in a manner known per se (cf. Sambrook et al., supra).
  • the transformed host cell is Echerichia coli.
  • the polypeptide When expressing the polypeptide in bacteria such as E. coli, the polypeptide may be retained in the cytoplasm, typically as insoluble granules (known as inclusion bodies), or may be directed to the periplasmic space by a bacterial secretion sequence. In the former case, the cells are lysed and the granules are recovered and denatured after which the polypeptide is refolded by diluting the denaturing agent. In the latter case, the polypeptide may be recovered from the periplasmic space by disrupting the cells, e.g. by sonication or osmotic shock, to release the contents of the periplasmic space and recovering the polypeptide.
  • sonication or osmotic shock to release the contents of the periplasmic space and recovering the polypeptide.
  • suitable mammalian cell lines are the COS (ATCC CRL 1650), BHK (ATCC CRL 1632, ATCC CCL 10), CHL (ATCC CCL39) or CHO (ATCC CCL 61) cell lines.
  • Methods of transfecting mammalian cells and expressing DNA sequences introduced in the cells are described in e.g. Kaufman and Sharp, J. MoL BjoL 159 (1982), 601 - 621 ; Southern and Berg, JL MoL Appl. Genet. 1 (1982), 327 - 341; Loyter et al., Proc. Natl. Acad. Sci.
  • yeasts cells include cells of Saccharomyces spp. or Schizosaccharomyces spp., in particular strains of Saccharomyces cerevisiae or Saccharomyces kluyveri. Methods for transforming yeast cells with heterologous DNA and producing heterologous polypeptides therefrom are described, e.g. in US 4,599,311, US 4,931,373, US 4,870,008, 5,037,743, and US 4,845,075, all of which are hereby incorporated by reference. Transformed cells are selected by a phenotype determined by a selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient, e.g. leucine.
  • a selectable marker commonly drug resistance or the ability to grow in the absence of a particular nutrient, e.g. leucine.
  • a preferred vector for use in yeast is the POT1 vector disclosed in US 4,931,373.
  • the DNA sequence encoding the polypeptide of the invention may be preceded by a signal sequence and optionally a leader sequence , e.g. as described above.
  • suitable yeast cells are strains of Saccharomyces sp., including Saccharomyces cerevisiae, Saccharomyces kluyveri, and Saccharomyces uvarum; Schizosaccharomyces pombe; Kluyveromyces sp., including Kluyveromyces lactis; Hansenula sp., including Hansenula polymorpha; Pichia sp., including Pichia pastoris, Pichia methanolica, and Pichia kluyveri; Yarrowia lipolytica; Candida sp., including Candida utilis, and Candida cacaoi; Geotrichum sp.; and Geotrichum fermentans (cf. Gleeson et al.,
  • Examples of other fungal cells are cells of filamentous fungi, e.g. Aspergillus spp., Neurospora spp., Fusarium spp. or Trichoderma spp., in particular strains of A. oryzae, A. nidulans or A. niger.
  • Aspergillus spp. for the expression of proteins is described in, e.g., EP 272 277, EP 230 023.
  • a filamentous fungus When a filamentous fungus is used as the host cell, it may be transformed with the DNA construct of the invention, conveniently by integrating the DNA construct in the host chromosome to obtain a recombinant host cell.
  • This integration is generally considered to be an advantage as the DNA sequence is more likely to be stably maintained in the cell. Integration of the DNA constructs into the host chromosome may be performed according to conventional methods, e.g. by homologous or heterologous recombination.
  • the transformed or transfected host cell described above is then cultured in a suitable nutrient medium under conditions permitting the expression of the present polypeptide, after which the resulting polypeptide is recovered from the culture.
  • the medium used to culture the cells may be any conventional medium suitable for growing the host cells, such as minimal or complex media containing appropriate supplements. Suit ⁇ able media are available from commercial suppliers or may be prepared according to published recipes (e.g. in catalogues of the American Type Culture Collection).
  • the polypeptide produced by the cells may then be recovered from the culture medium by conventional procedures including separating the host cells from the medium by centrifugation or filtration, precipitating the proteinaceous components of the supernatant or filtrate by means of a salt, e.g. ammonium sulphate, purification by a variety of chromatographic procedures, e.g. ion exchange chromatography, gelfiltration chromatography, affinity chromatography, or the like, dependent on the type of polypeptide in question.
  • a salt e.g. ammonium sulphate
  • reaction was repeated with additionally 25 mg NaBH 4 and 5 times 25 ml 37% formalin during a 55 minutes period. (200 ml reaction No.2 sample was taken). To quench the reaction, the main part of the reaction mixture was adjusted to pH 6.3 with 1 M acetic acid.
  • the lysine in position 30 of the mature enzyme was substituted with an arginine using the method "Splicing by overlap extension", Horton et al., Gene 77, 1989, pp. 61-68. Sequence positions in the following is taken from Ohara et al., J.Biol.Chem. 264 (34), 1989, pp. 20625- 20631.
  • the two PCR products were mixed and denatured. Taq polymerase was added. The product was reannealed at 50°C and extended for 4 minutes at 72°C. The primers MHJ 3989 and 3990 were added and the DNA was amplified for 20 cycles.
  • the resulting PCR product was cut with Asc I and Xho I and cloned into pSX547, where the wild type Asc I - Xho I fragment had been removed.
  • the resulting plasmid pSX582 was transformed into E.coli 3110 lac l q ; the resulting strain was plated onto LB-plates with 200 mg/ml ampicillin (J.H. Miller (1972), Experiments in Molecular genetics, Cold Spring Harbor Laboratory).
  • the resulting strain was grown in liquid LB medium containing 0.4% lactose for 44 hours at 26mC whereafter the culture was centrifuged and the supernatant tested for lysyl- endopeptidase activity with Benzoyl-lysyl-pNA. The result was positive. The mutant enzyme was recovered from the supernatant.
  • API with lysines at position 30 and 49 mutated to arginine API with lysines at position 30 and 49 mutated to arginine:
  • the mutant described in example 1 was used as template, resulting in a double mutant with both lysines in position 30 and 49 substituted with arginines.
  • the lysine in position 49 of the mature enzyme was substituted with an arginine using Stratagenes "QuickChangeTM Site-Directed Mutagenesis Kit”. Sequence positions in the following is taken from Ohara et al.. J.Biol.Chem. 264 (34), 1989, pp. 20625-20631.
  • Primer EliA 9 and 10 Two complementary primers going from position 1006-1037 were made (primer EliA 9 and 10). These primers overlap with the codon for amino acid 49, changing this from AAG (lysine) to CGT (arginine).
  • Primers EliA9 and 10 were mixed with the template plasmid pSX582. Pfu polymerase was added and and the mixture denatured. Subsequently 18 cycles were performed as follows: denature 45 seconds at 97°C, anneal 1 minute at 55°C and extend 8 min at 68°C. Finally the mixture was digested with Dpn1 for 1 hour at 37°C to remove template (pSX582).
  • the resulting double stranded nicked plasmids were transformed into E.coli and plated onto LB-plates with 200 ⁇ g/ ml ampicillin. Colonies were picked and a plasmid containing the desired mutation was identified by DNA sequencing. This was named pEA186.
  • the resulting strain was grown at 26°C in liquid LB medium to an OD600 of 1.5. Subsequently 0.4% lactose was added and the growth continued for 44 hours whereafter the culture was centrifuged and the supernatant tested for lysyl-endopeptidase activity with Benzoyl- lysyl-pNA. The result was positive. The mutant enzyme was recovered from the supernatant.
  • EliA 9 5'-CCG CCA ACG ACC GCC GTA TGT ACT TCC TGA CC-3"
  • EliA 10 5'-GGT CAG GAA GTA CAT ACG GCG GTC GTT GGC GG-5"
  • API with lysines at position 30 and 106 mutated to arginine API with lysines at position 30 and 106 mutated to arginine:
  • the mutant described in example 1 was used as template, resulting in a double mutant with both lysines in position 30 and 106 substituted with arginines.
  • the lysine in position 106 of the mature enzyme was substituted with an arginine using Stratagenes "QuickChangeTM Site-Directed Mutagenesis Kit".
  • the mutation was introduced using the method described in Example 3, except that the complementary primers are going from position 851 to 882 (primer EliA 11 and 12). These primers overlap with the codon for amino acid 106, changing this from AAG (lysine) to CGT (arginine).
  • the resulting plasmid was named pEA187.
  • the supernatant from the resulting culture was tested for API activity and found to be positive as described in example
  • EliA 11 5 -CGG GTT CGA CGG TCC GTG CGA CCT ACG CCA CC-3'
  • EliA 12 5'-GGT GGC GTA GGT CGC ACG GAC CGT CGA ACC CG-3'
  • API with lysines at position 30, 46 and 106 mutated to arginine API with lysines at position 30, 46 and 106 mutated to arginine:
  • Plasmid pEA187 was cut with restriction enzymes Pstl and BspMI. This results in a DNA fragment of 0.4 kb containing the code for arginine at position 106 of API. Plasmid pEA186 was cut with the same restriction enzymes. A DNA fragment of 3.8 kb was isolated and ligated with the 0,4 kb fragment, resulting in a plasmid pEA189 encoding an API where lysines at position 30, 49 and 106 has been mutated to arginine. The resulting plasmid was named pEA189.
  • ELISA plate 96 wells
  • ELISA reader e.g. Bio-TekTM EL 340
  • a stock substrate solution containing 0.004 M Z-Lys-pNA in 0.1 M tris/HCI buffer, pH 8.0 is prepared by dissolution of 100 mg Z-Lys-pNA,HCI in 1 ml of DMSO and subsequent dilution to 57 ml with tris/HCI buffer. The pH value in the resulting solution is readjusted to 8.0.
  • Standard enzyme solution 1.0 ml of water is added to a vial containing 10 units of lyophilized API (WAKO Pure
  • Test enzyme solution The enzyme solution to be tested is suitably diluted with tris/HCI buffer, pH 8.0.
  • COMPUTER READABLE FORM (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • FRAGMENT TYPE internal
  • Xaa in pos.30 represents the amino acid 6-N-dimethyllysine.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

Nouveaux variants de protéase I d'Achromobacter lyticus, dans lesquels un ou plusieurs restes lysine au niveau des positions 30, 49, 106, 155 et 203 ont été remplacés par un autre reste aminoacide qui peut être codé par des acides nucléiques de recombinaison, ou dans lesquels un ou plusieurs restes lysine au niveau des positions précitées ou d'autres restes aminoacide introduits au niveau desdites positions ont été chimiquement modifiés. L'invention se rapporte en outre à des acides nucléiques de recombinaison codant lesdits variants de protéase, ainsi qu'à des vecteurs et cellules hôtes comprenant lesdits produits de recombinaison. Ces variants de protéase sont utiles pour le clivage des liaisons peptidiques Lys-X dans des polypeptides, tels que les préproinsulines.
PCT/DK1997/000100 1996-03-12 1997-03-07 Nouveaux variants de protease d'achromobacter lyticus WO1997033984A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU20913/97A AU2091397A (en) 1996-03-12 1997-03-07 Novel achromobacter lyticus protease variants

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DK28496 1996-03-12
DK0284/96 1996-03-12

Publications (1)

Publication Number Publication Date
WO1997033984A1 true WO1997033984A1 (fr) 1997-09-18

Family

ID=8091832

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/DK1997/000100 WO1997033984A1 (fr) 1996-03-12 1997-03-07 Nouveaux variants de protease d'achromobacter lyticus

Country Status (3)

Country Link
AU (1) AU2091397A (fr)
WO (1) WO1997033984A1 (fr)
ZA (1) ZA972083B (fr)

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6171823B1 (en) * 1994-12-09 2001-01-09 Novo Nordisk A/S Process for producing extracellular proteins in bacteria
US6190883B1 (en) 1998-09-09 2001-02-20 Novo Nordisk A/S Method for the production of heterologous polypeptides in transformed yeast cells
DE10106541A1 (de) * 2001-02-13 2002-08-22 Schill & Seilacher Enzympräparat und dessen Verwendung in der Gerberei
US6753155B1 (en) * 1997-05-13 2004-06-22 The United States Of America As Represented By The Secretary Of The Army Protein biomarker for mustard chemical injury
US8465959B2 (en) * 2003-06-19 2013-06-18 Novozymes A/S Proteases and methods for producing them
CN112824527A (zh) * 2019-11-20 2021-05-21 珠海联邦制药股份有限公司 人工设计的赖氨酰内切酶及编码序列和发酵方法
CN115717137A (zh) * 2022-12-27 2023-02-28 北京惠之衡生物科技有限公司 赖氨酰特异性内切酶突变体及其制备方法和应用
WO2024036099A1 (fr) * 2022-08-08 2024-02-15 Waters Technologies Corporation Protéases modifiées ayant une résistance à l'autolyse améliorée

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0200451A1 (fr) * 1985-04-22 1986-11-05 Genentech, Inc. Composition d'urokinase résistant aux protéinases, sa production et son utilisation
EP0201153A2 (fr) * 1985-02-09 1986-11-12 Beecham Group Plc Enzyme modifié et son procédé de préparation
EP0367302A2 (fr) * 1982-04-23 1990-05-09 Wako Pure Chemical Industries, Ltd. Procédé pour la semi-synthèse d'insuline humaine protéase I d'Achromobacter réticulée soluble dans l'eau pour sa mise en oeuvre et procédé pour la préparation de celle-ci
EP0387646A1 (fr) * 1989-03-14 1990-09-19 Wako Pure Chemical Industries Ltd Gène de la protéase I de Achromobacter et produit de ce gène

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0367302A2 (fr) * 1982-04-23 1990-05-09 Wako Pure Chemical Industries, Ltd. Procédé pour la semi-synthèse d'insuline humaine protéase I d'Achromobacter réticulée soluble dans l'eau pour sa mise en oeuvre et procédé pour la préparation de celle-ci
EP0201153A2 (fr) * 1985-02-09 1986-11-12 Beecham Group Plc Enzyme modifié et son procédé de préparation
EP0200451A1 (fr) * 1985-04-22 1986-11-05 Genentech, Inc. Composition d'urokinase résistant aux protéinases, sa production et son utilisation
EP0387646A1 (fr) * 1989-03-14 1990-09-19 Wako Pure Chemical Industries Ltd Gène de la protéase I de Achromobacter et produit de ce gène

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6171823B1 (en) * 1994-12-09 2001-01-09 Novo Nordisk A/S Process for producing extracellular proteins in bacteria
US6753155B1 (en) * 1997-05-13 2004-06-22 The United States Of America As Represented By The Secretary Of The Army Protein biomarker for mustard chemical injury
US6190883B1 (en) 1998-09-09 2001-02-20 Novo Nordisk A/S Method for the production of heterologous polypeptides in transformed yeast cells
DE10106541A1 (de) * 2001-02-13 2002-08-22 Schill & Seilacher Enzympräparat und dessen Verwendung in der Gerberei
DE10106541B4 (de) * 2001-02-13 2005-05-12 Schill + Seilacher Aktiengesellschaft Enzympräparat und dessen Verwendung in der Gerberei
US8465959B2 (en) * 2003-06-19 2013-06-18 Novozymes A/S Proteases and methods for producing them
CN112824527A (zh) * 2019-11-20 2021-05-21 珠海联邦制药股份有限公司 人工设计的赖氨酰内切酶及编码序列和发酵方法
CN112824527B (zh) * 2019-11-20 2023-05-26 珠海联邦制药股份有限公司 人工设计的赖氨酰内切酶及编码序列和发酵方法
CN116334050A (zh) * 2019-11-20 2023-06-27 珠海联邦制药股份有限公司 人工设计的赖氨酰内切酶及编码序列和发酵方法
CN116334050B (zh) * 2019-11-20 2024-02-02 珠海联邦制药股份有限公司 人工设计的赖氨酰内切酶及编码序列和发酵方法
WO2024036099A1 (fr) * 2022-08-08 2024-02-15 Waters Technologies Corporation Protéases modifiées ayant une résistance à l'autolyse améliorée
CN115717137A (zh) * 2022-12-27 2023-02-28 北京惠之衡生物科技有限公司 赖氨酰特异性内切酶突变体及其制备方法和应用
CN115717137B (zh) * 2022-12-27 2024-01-26 北京惠之衡生物科技有限公司 赖氨酰特异性内切酶突变体及其制备方法和应用

Also Published As

Publication number Publication date
ZA972083B (en) 1997-09-28
AU2091397A (en) 1997-10-01

Similar Documents

Publication Publication Date Title
Collins-Racie et al. Production of recombinant bovine enterokinase catalytic subunit in Escherichia coli using the novel secretory fusion partner DsbA
US5707826A (en) Enzymatic method for modification of recombinant polypeptides
KR0157983B1 (ko) 아미드화 효소 발현계
CA2153254C (fr) Clonage d'enterokinase et methode d'utilisation
EP2507258B1 (fr) Nouvelles peptidyl a-hydroxyglycine a-amide lyases
AU740742B2 (en) Modified carboxypeptidase
WO2011056911A9 (fr) Compositions et procédés permettant d'améliorer la production d'une substance biologique compositions and methods for enhancing production of a biological product
Sagiya et al. Direct high-level secretion into the culture medium of tuna growth hormone in biologically active form by Bacillus brevis
JPH04166085A (ja) 新規プロテアーゼ
WO1997033984A1 (fr) Nouveaux variants de protease d'achromobacter lyticus
EP0647710B1 (fr) Nouvelle protease
EP1634954B1 (fr) Protease, adn codant pour cette protease et procede de production de cette protease
KR0161656B1 (ko) 글루카곤의 생산방법
CA2486195C (fr) Carboxypeptidase b exprimee par ecombinaison et sa purification
JP2000501617A (ja) 酵母細胞におけるｎ末端を伸長されたタンパクの発現のためのベクター
KR100714116B1 (ko) 췌장의 프로카복시펩티다제 ｂ를 사용한 인슐린의 제조
JP3503319B2 (ja) 新規チオールプロテアーゼをコードするｄｎａおよびこれを用いた該チオールプロテアーゼの製造方法
US6428997B1 (en) Aminopeptidase derived from Bacillus licheniformis and process for preparation of natural type proteins
Lien et al. Linkers for improved cleavage of fusion proteins with an engineered α‐lytic protease
EP0718403A1 (fr) Procédé pour la production de matrilysine au moyen d'ADN recombinant
EP1538203A1 (fr) Carboxypeptidase B recombinante et sa purification
JP2003079379A (ja) 成長ホルモンの高発現用ｄｎａおよびその使用
US20040253703A1 (en) Novel aminopeptidase derived from bacilius licheniformis, gene encoding the aminopeptidase, expression vector containing the gene, transformant and method for preparation thereof
EP1421102A2 (fr) Procede pour produire des polypeptides acyles
WO2003010287A1 (fr) Immobilisation de keratinase dans la proteolyse et la keratinolyse

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GE GH HU IL IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK TJ TM TR TT UA UG US UZ VN YU AM AZ BY KG KZ MD RU TJ TM

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH KE LS MW SD SZ UG AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
NENP Non-entry into the national phase

Ref country code: JP

Ref document number: 97532204

Format of ref document f/p: F

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

NENP Non-entry into the national phase

Ref country code: CA

122 Ep: pct application non-entry in european phase