WO2000014259A1 - Souches de candida boidinii presentant une activite protease reduite et utilisation de ces dernieres en tant qu'hote pour produire une proteine exogene - Google Patents

Souches de candida boidinii presentant une activite protease reduite et utilisation de ces dernieres en tant qu'hote pour produire une proteine exogene Download PDF

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WO2000014259A1
WO2000014259A1 PCT/JP1999/004802 JP9904802W WO0014259A1 WO 2000014259 A1 WO2000014259 A1 WO 2000014259A1 JP 9904802 W JP9904802 W JP 9904802W WO 0014259 A1 WO0014259 A1 WO 0014259A1
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strain
gene
candida
seq
proteinase
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PCT/JP1999/004802
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Toshihiro Komeda
Keiji Kondo
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Kirin Beer Kabushiki Kaisha
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
    • C07K14/40—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts from Candida
    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • C12N1/165—Yeast isolates
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/67—General methods for enhancing the expression
    • 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)
    • C12N9/58—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi
    • C12N9/60—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi from yeast
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/72—Candida

Definitions

  • the present invention relates to a Candida boidini O protease gene, a Candida boidini strain having a modified DNA of the protease gene, and a method for producing a heterologous protein using the Candida boidini strain as a host. If a protein expression system using this strain of Candida boizini as a host can be used, the desired heterologous protein can be produced with high yield.
  • the present invention also relates to a signal peptide useful for the secretory expression of a heterologous protein using Candida 'boizini as a host, a secretory expression system for a heterologous protein using the signal peptide, and a heterologous protein using the secretory expression system. A method for producing the same.
  • Candida boidii has been developed as an effective host for heterologous protein expression systems.
  • Alcohol oxidase, dihydroxyacetone synthase, and formate dehydrogenase present in the methanol utilization pathway are produced in significant quantities when cultured in the presence of methanol, and a method for expressing heterologous genes using the regulatory regions of these genes has been developed. It has been studied (Japanese Patent Application Laid-Open No. Hei 5-3444895, International Publication No. W097110345).
  • the target product may be degraded by a protease derived from a host. In such a case, the production amount of the target protein decreases, and the purification of the target protein becomes difficult due to contamination with protein degradation products.
  • a culture method that inhibits the protease activity that degrades the target protein has been used. For example, by adjusting the pH of the culture medium It is possible to inhibit the protease action.
  • this method will affect the growth of host yeast expressing certain heterologous proteins, and is only effective for extracellular protein degradation.
  • Proteinase A and proteinase B are proteinases localized in the vacuole, and are encoded by the _EP4 gene and 1 gene, respectively.
  • Studies of the yeast S acchar 0 my cescerevisiae show that proteinase A and proteinase B activate themselves and other proteases such as carboxypeptidase Y (vanden Hazel, HB et al., YEAST , ⁇ 2_, 1 (1996)).
  • S acchar omy cescerevisiae and Picnia pastoris and Candida boidii are essentially mycologically distinct, with many metabolic and physiological differences. Therefore, it is easily speculated that the proteolytic mechanism is different.
  • S acchar omy cescerevisiae ⁇ Pichiapastoris has completely lost activity due to the proteinase A gene deletion. Loss of carboxypeptidase Y is about 4 in Candida boizini, which lacks the proteinase A gene. Although 0% activity was found to remain, this example also shows that the proteolytic mechanism of Candida voizi differs from that of the other two yeasts.
  • Protease-deficient strains are obtained by screening mutant strains and disrupting genes. In order to screen for mutants, a huge number of mutants must be analyzed. Unlike Sacc ⁇ ar ojny cescerevisiae, Pichiapastor J's, Candida voidii, which has no sporulation ability, It is impossible to analyze whether mutations have been introduced only into the target gene by crossover. In addition, revertants in which the mutated trait returns to the state before the mutation may occur. On the other hand, the gene disruption method is an effective technique because it is possible to delete only the target gene, but the target gene: region of the host must be obtained in order to perform the gene disruption. However, no knowledge of such proteases and their genes for Candida boidii has been obtained to date.
  • An object of the present invention is to provide a protease derived from Candida boidii, a DNA having a nucleotide sequence encoding the protease, and a strain of Candida boidini lacking the protease gene. I do. It is also an object of the present invention to provide a signal peptide useful for a yeast secretion expression system comprising a DNA sequence encoding a secretory signal peptide derived from the protease protein, and a method for secretory production of a heterologous protein using the signal peptide. With the goal. Summary of the Invention
  • the present inventors analyzed the protease gene of the methanol-assimilating yeast Ca / 'boich'nii and conducted intensive studies to achieve efficient expression of the heterologous gene.
  • the protein gene deletion Candida ⁇ The present invention has been completed by achieving high expression of a heterologous gene using a Boijini strain.
  • the present invention provides a Candida boizini strain with reduced protease activity.
  • the protease is preferably one that is involved in the degradation of a heterologous protein produced by expression by recombinant technology. By losing at least one activity of such a protease, overall protease activity can be reduced.
  • the present invention provides a Candida boizini strain which has lost proteinase 8, proteinase B or both proteinase activities. More specifically, such strains are Candida boizini SK740, SK741, SK774 or SK775 strains.
  • the present invention also provides for transforming the above-mentioned Candida 'boizini strain with an expression vector containing a gene encoding a useful heterologous protein, culturing it in an appropriate medium, and recovering the produced heterologous protein. And a method for producing a protein.
  • the present invention provides a method for transforming Candida's Boizini SK741 strain with an expression vector containing a gene encoding cathepsin C, culturing it in an appropriate medium, and producing the produced cathepsin C.
  • a method for producing cathepsin C including recovering.
  • the expression vector can include DNA encoding a secretory signal peptide sequence adjacent to the 5 'end of the gene encoding the heterologous protein.
  • Signal peptides have a role in transporting precursor proteins synthesized by ribosomes to the membrane.They are cleaved by sidinal peptides, resulting in the secretion of mature proteins outside the cell. Become.
  • the secretory signal peptide sequence consists of the amino acid sequence shown in SEQ ID NO: 4 derived from proteinase protein, more specifically from Candida boizii proteinase A.
  • the present invention further relates to the amino acid sequence at positions 23 to 420 shown in SEQ ID NO: 2, or at least 80%, preferably at least 90%, more preferably at least 95% homology in the sequence.
  • a proteinase A or a derivative thereof derived from a Candida boizini strain having an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added so as to have protease activity, and having protease activity.
  • the present invention further provides a DNA encoding proteinase A or a derivative thereof derived from the above Candida's Boizini strain.
  • the present invention also relates to the amino acid sequence shown in SEQ ID NO: 2, or one or more amino acids having a homology of at least 80%, preferably at least 90%, more preferably at least 95% in the sequence. It is provided a precursor proteinase A or a derivative thereof derived from Candida's Boizini strain, which has an amino acid sequence having substitution, insertion and / or addition.
  • the present invention further provides a DNA encoding the above-described precursor proteinase A derived from Candida's Boizini strain or a derivative thereof.
  • the DNA can have the base sequence shown in SEQ ID NO: 3.
  • the present invention also provides the amino acid sequence shown in SEQ ID NO: 5, or one or more amino acids having at least 80%, preferably at least 90%, more preferably at least 95% homology in the sequence.
  • a proteinase B or a derivative thereof derived from Candida bovis two strains having an amino acid sequence in which the amino acid sequence has been substituted, substituted, or added or has protease activity.
  • the present invention further provides a DNA encoding proteinase B or a derivative thereof derived from the above Candida boizini strain. Specifically, the DNA has the base sequence shown in SEQ ID NO: 6.
  • the proteinases A and B of the N. boizini strain or the precursor proteinases A and B and the DNAs encoding them are proteinases B and B of the baker's yeast accharomyces cerevisiae) or Pichia pastoris ichia pastor is). It has sequence similarity to the PEP4 and PRB1 genes that encode them, but Both Haccharomyces cerevisiae and Pichia pastoris have a homology of less than 80% and are essentially different from the PG A gene and the PRB1 gene.
  • the present invention The above-mentioned ⁇ derivative '' in the above, as long as the desired protease activity is obtained, and / or the sequence shown in SEQ ID NO: 2, 3 or SEQ ID NO: 5, 6 and at least 80%, preferably Substitutions, as long as they have at least 90%, more preferably at least 95%, homology. It means that mutations such as deletion, insertion or addition may be included.
  • the derivative of the present invention is useful for destroying DNA having any nucleotide sequence encoding an amino acid sequence essentially identical to the amino acid sequence shown in SEQ ID NO: 2 or 5, or a homologous gene on a chromosome. DNA sequences with sufficient homology are also included.
  • the derivative of the present invention substantially comprises the amino acid sequence or the base sequence shown in SEQ ID NO: 2, 3, 5 or 6 in both the protein and the DNA (or, substantially, It can be expressed as having an array.
  • the present invention also provides a secreted synaldal peptide of Candida's bodini derived proteinase A, comprising the amino acid sequence shown in SEQ ID NO: 4.
  • FIG. 1 shows the restriction map of the plasmid pCPRA1 containing the proteinase A gene.
  • FIG. 2 shows the procedure for constructing the proteinase A gene disruption plasmid pDPRA1.
  • FIG. 3 shows a restriction enzyme map of the P ⁇ sodium 4 locus of Candi daboidiniini SK612, SK740, and SK741 strains.
  • Figure 4 shows a restriction map of the proteinase B gene and the plasmids pCPRB1 and pCPRB2.
  • FIG. 5 shows the structure of the proteinase B gene disruption plasmid pDPRB1.
  • FIG. 6 shows a restriction enzyme map of the PRB1 locus of Candidaboidiniini SK741, SK774, and SK775.
  • FIG. 7 shows the procedure for constructing plasmid p CTC—S1.
  • FIG. 8 shows the procedure for constructing plasmid pECTC-S1.
  • FIG. 9 shows the strains of Candidaboidinii SK612 and SK741.
  • 5 shows the force-tepsin C activity of the culture supernatant of a cathepsin C-expressing strain using E. coli as a host.
  • strains include two strains of Pseudomonas boidi in which the PEP4 gene encoding wild-type proteinase A has been replaced with the modified Society4 gene as described above. Not only does not produce wild-type proteinase A at all, but also significantly inhibits the activities of proteases such as riboxipeptidase Y and proteinase B activated by proteinase A. It is also possible to produce a Candida boizini strain in which, in addition to the P £ P4 gene, the B1 gene encoding proteinase B has been replaced with the PSl gene modified as described above.
  • One gene-deleted strain is characterized in that it has the same growth ability as a wild type under culture conditions using a nutrient medium. This means that the presence or absence of these genes does not affect the growth of Candida boidii under nutritional conditions. Therefore, the yeast with suppressed protease activity according to the present invention is an excellent host for heterologous protein production. In particular, the yeast can efficiently produce a protease-sensitive heterologous protein.
  • a transformant obtained by transforming the above-described two strains of the present Candida boili strain with an expression vector containing a gene encoding a heterologous protein is further cultured.
  • a method for producing a heterologous protein is further cultured.
  • a heterologous gene means any gene to be expressed, for example, forceepsin C, epidermal growth factor (EGF), insulin-like growth factor 1 (IGF-1), human serum albumin, erythropoietin (EPO), Thrombopoietin (TPO) and the like, but are not limited thereto.
  • the heterologous gene may be obtained by any technique.
  • the expression vector can contain DNA encoding a signal peptide sequence for secretion of the heterologous protein, adjacent to (ie, flanking) the fifth end of the gene encoding the heterologous protein.
  • the heterologous protein produced by the expression can be secreted out of the cell.
  • the secretory signal peptide for example, a signal sequence originally possessed by cathepsin C, a secretory signal peptide sequence of ⁇ factor of baker's yeast, and a Cariaida boizini proteinase sidanal peptide can be used.
  • examples of the heterologous gene include a gene encoding cathepsin C, which is a peptidyl protease from Japan.
  • This enzyme is a protease that degrades two amino acids at a time from the N-terminus of the protein, and is an industrially useful enzyme.
  • This novel gene is characterized as a protein having an amino acid sequence substantially identical to the amino acid sequence shown in SEQ ID NO: 8.
  • Cathepsin C is expressed as an inactive precursor (prebu-oral body), and subsequent processing cleaves the peptide region that functions as a secretory signal, followed by cleavage of the propeptide ′ region, resulting in an active mature protein. become.
  • a peptide region is generally a peptide fragment contained in an inactive precursor of a protein, and the peptide is cleaved and removed from the inactive precursor by a specific protease or the like, and the activity specific to the protein is obtained. Is shown. Therefore, the propeptide sequence of dicatehepsin C followed by the polypeptide sequence constituting the mature protein is necessary for activation of the enzyme during secretion. For this reason, secretory expression of the enzyme in yeast requires the addition of a secretinous sequence to the N-terminal of the cathepsin C propeptide.
  • the secretory signal peptide those exemplified above, preferably Candida- The signal peptide of Boise II proteinase A can be used.
  • the above protease gene-deficient strain was particularly useful for expression of the protein.
  • the present invention relates to a Candida 'boizini strain producing cathepsin C, preferably a Candida' boizini strain having reduced protease activity, and a Candida 'boizini strain having reduced protease activity.
  • a method for producing cathepsin C is also included.
  • the present inventors have solved the above-mentioned problems by (1) elucidating the nucleotide sequences of the proteinase A and proteinase B genes of Candida 'boizii, and (2) constructing a plasmid for disrupting the proteinase gene. (3) A transformant was prepared using this plasmid to obtain a Candida 'boizini strain lacking the protease gene. Furthermore, (4) it was confirmed that when a heterologous gene was expressed using a Candida boizini strain lacking the protease gene as a host, the production amount was superior to that obtained when a wild-type strain was used as a host. It has been completed.
  • Candida'boizini AT CC48180 strain is exemplified.
  • the cloning step can be performed according to a known method (Molecular Cloning (1989), Methods in Enzymology 194 (1991)).
  • a gene transfer vector incorporating a DNA fragment derived from the total DNA of the yeast or a cDNA fragment resynthesized from the mRNA of the yeast is introduced into a host, and the yeast gene Make a rally.
  • B Then, a desired clone is selected from the gene library, and the above-mentioned cloning step can be performed by amplifying the clone.
  • Extraction of total yeast DNA is performed, for example, by preparing yeast protoplasts, removing the cell debris from the protoplasts by a generally known DNA extraction method, and then subjecting the DNA to alcohol precipitation under a high salt concentration, followed by phenol Or after extraction of form And purification by alcohol precipitation.
  • DNA can be extracted by cell disruption using glass beads or the like, but it is easy to prepare high molecular weight DNA. It is preferred to carry out the above protoplast method.
  • the obtained chromosomal DNA is digested with an appropriate restriction enzyme, ligated to an appropriate vector, and then transformed into an appropriate E. coli host, whereby a genomic library can be obtained.
  • vector used in this case examples include commercially available plasmids such as pBR strain, pUC strain, and Bluescript strain, which are generally known as known gene library preparing vectors. Can also be used. Further, phage vectors or cosmids of the gt system or the EMBL system can be widely used.
  • a host corresponding to the type of the above vector can be used.
  • clones having the desired proteinase A and proteinase B genes were colonized using a labeled probe containing a sequence specific to each gene. Hybridization method, plaque hybridization, etc. Can be selected and acquired.
  • the sequences specific to the proteinase A and proteinase B genes used for the probe were designed based on the amino acid sequences conserved by proteases of various origins, and the primers based on the codon usage of Candida bodily.
  • the desired DNA fragment is obtained by specifically amplifying the DNA fragment by the PCR method using the Candida voidii chromosome DNA as type II.
  • two sets of oligonucleotides corresponding to the amino acid sequence of the protease purified from Candida boidii were synthesized, and the primers were used as primers to carry out desired PCR by using the chromosomal DNA of Candida boidii as a type II. It is also possible to specifically amplify and obtain a DNA fragment to be obtained.
  • the synthesized oligonucleotide can also be used as a probe.
  • the determination and confirmation of the nucleotide sequence of the desired gene obtained by the above-mentioned method can be performed, for example, by the Maxam-Gilbert method in Enzymology (65, 499 (1980)). It can be performed by the dideoxynucleotide chain termination method (Messing, J. and Vieire, J., Gene, Id, 269 (1982)) and its automated modification.
  • the DNA encoding the protease was modified so that it could not produce a functional protease protein and was inserted into an appropriate vector along with DNA, a selection marker gene, etc., as a gene disruption plasmid. used. By site-specific integration using the present plasmid, the gene can be prepared by replacing the gene on the chromosome.
  • a DNA sequence that has been modified so that a functional protease protein cannot be produced refers to a DNA sequence encoding a protein in which the bases have been substituted, a portion of the DNA sequence has been deleted, or at least one nucleotide has been deleted.
  • such a modified DNA sequence can be prepared by inserting a transformation marker gene or the like into a DNA sequence encoding a protein. It is said that such a DNA fragment can be used to destroy a gene on the chromosome and to screen for a mutant having a modified protease gene using the introduced transforming gene as an index.
  • the selectable marker gene used here include an antimicrobial resistance gene such as G418 and a gene complementary to the auxotrophy of the host such as URA3 and LEU2. Insertion of the components of the expression vector into the vector can be easily performed by those skilled in the art by referring to the description in the Examples below or by a conventional technique.
  • the Candida boizini strain whose protease activity was suppressed as compared to the wild-type strain, It can be produced by transforming a suitable yeast host with a DNA sequence modified so that the above-mentioned functional protease protein cannot be produced, and replacing an endogenous gene.
  • a suitable yeast host with a DNA sequence modified so that the above-mentioned functional protease protein cannot be produced, and replacing an endogenous gene.
  • the target gene on the chromosome is physically removed by gene replacement to replace the target gene with the modified gene.
  • This is a linear DNA fragment having a terminal sequence homologous to the 5 ′ and 3 ′ regions of the target gene, preferably a modified DN containing a gene disrupted by insertion of a transformation marker gene or the like. This is achieved by transforming a yeast host with the A fragment.
  • a modified human A3 gene which can be removed from the chromosome by spontaneous recombination can be used.
  • the modified human A3 gene has a structure in which DNA sequences homologous to its 5 'and 3' sides are arranged in the same direction. This makes it possible for spontaneous recombination between these repetitive sequences to occur after integration on the yeast chromosome, thereby causing the A3 gene to drop out. Can be reused.
  • the U ra- strain becomes resistant to 5-fluoroorotic acid (5-FOA)
  • the sporadic homologous recombination of the A3 gene from among the F ra- It is easy to select Ura-strains that have dropped out.
  • a method also called a pop-in pop-art may be used. It consists of a part of the endogenous target gene generated after the transformation after introducing the plasmid DNA containing the modified gene into the target locus by homologous exchange and a part of the modified gene used for the transformation.
  • a functional gene is removed by spontaneous homologous recombination between two genes, and a strain in which the modified gene remains is selected.
  • the URA3 gene dropped out of the 5-F ⁇ A-sensitive Ura + strain by spontaneous homologous recombination. Selection of strains is easy.
  • a protoplast method lithium acetate, an electric pulse method, or the like can be used.
  • the strain of Candida boizini used for the transformation but ATCC 48180 strain, IF 0 100 35 strains and the like are exemplified.
  • the strain is preferably a strain in which at least one auxotrophic marker gene has been deleted, and examples thereof include an A3 gene deleted strain and a gene deleted strain.
  • the heterologous gene is obtained by inserting a heterologous gene expression vector inserted into an appropriate vector together with a promoter sequence, a structural gene of a heterologous protein, an expression unit having a terminator sequence and a selection marker gene in the direction of the reading frame of transcription. It is performed by introduction into an appropriate host cell.
  • the prosthesis gene is disrupted as described above, followed by transformation with DNA encoding a heterologous protein.
  • Examples of a promoter for expressing a recombinant heterologous protein include a Candida boizini alcoholoxidase gene promoter (Japanese Patent Application Laid-Open No. 5-344895) and a formate dehydrogenase gene promoter (International Publication No. WO97 / 10345). ) Etc. are exemplified.
  • Examples of the terminator include terminator of Candida albicans, which is the origin of Candida albicans (Japanese Patent Application Laid-Open No. 5-344895), terminator of formate dehydrogenase gene, terminator of actin gene (International Publication No. W097 / 10345) and the like.
  • a signal sequence for secretion to the N-terminal of the heterologous protein, secretion of the heterologous protein becomes possible.
  • a secretory signal peptide sequence of a factor of baker's yeast (5. cerevisiae) and the like can be used in addition to the secretinous peptide sequence of proteinase A provided in the present invention.
  • the expression vector may be integrated into the host chromosomal DNA or may be present in a plasmid state using a vector having an autonomously replicating sequence capable of self-replication in the host cell.
  • the copy number of the heterologous gene present in the host cell may be one copy or plural.
  • the target gene expression product can be obtained by culturing the transformant thus obtained and purifying it from the resulting culture.
  • the medium may include one or more carbon sources such as methanol, glycerol, and glucose, and one or more nitrogen sources such as yeast extract, tryptone, meat extract, peptone, casamino acid, and ammonium salt, and phosphate, sodium, and potassium.
  • carbon sources such as methanol, glycerol, and glucose
  • nitrogen sources such as yeast extract, tryptone, meat extract, peptone, casamino acid, and ammonium salt, and phosphate, sodium, and potassium.
  • Inorganic salts such as magnesium, magnesium, potassium, iron, copper, manganese, and cobalt are added, and if necessary, trace nutrients such as various vitamins, amino acids, and nucleotides are added as needed.
  • the pH of the medium is preferably in the range of 5-8.
  • the culture temperature is usually 15 to 37 ° C, preferably around 28 ° C.
  • the culturing time is about 24 to 1000 hours, and the culturing can be carried out by batch culturing under standing, shaking, stirring, aeration or continuous culturing.
  • a normal protein purification means can be used to collect the gene product from the culture. For example, when produced in a transformed cell, a crude protein solution containing a gene product is obtained by sonication, trituration, pressure crushing, etc. of the bacterial cells by a conventional method. If necessary, a protease inhibitor is added. When produced in the culture supernatant, the gene product can be recovered from the culture solution itself. The resulting solution is filtered, centrifuged or the like to remove the solid portion, and a crude protein solution is obtained. If necessary, nucleic acid is removed by reprotamine treatment or the like.
  • a Ca 73 dida 5 oj 'd /: strain having reduced protease (or proteolytic) activity is provided.
  • the yield can be improved.
  • Example Examples will be given below to further specifically explain the present invention, but the present invention is not limited thereto.
  • Non-yeast Sacharomyces cerevisiae
  • Pichia pastoris Tokuhei 6-506117
  • PRA3 5 '-AT AC AWGAWAC TT C YAAWGT RT AATC R TAWGG—3' (SEQ ID NO: 13).
  • Primer PRA 5 corresponds to the amino acid sequence DFAEAT SEPGL (SEQ ID NO: 10), and primer PRA 3 is the sequence of the complementary strand of the base sequence corresponding to the amino acid sequence PYDYTLEV SGSCI (SEQ ID NO: 11).
  • the nucleotide sequence was found to be highly homologous to the amino acid sequence of Sacc7: aroycescerevisiae and the Soci7 "s4 gene derived from Pichia.
  • the DNA fragment was identified as a part of the 4 genes of C andidaboidinii, since a nucleotide sequence encoding an amino acid sequence having a similarity was identified.
  • the chromosome DNA of Candidab boidinii ATCC48180 strain was digested with various restriction enzymes and subjected to 0.8% agarose gel electrophoresis.
  • the isolated DNA was transferred using HybondN + nylon membrane (Amersham).
  • the DNA fragment obtained in Example (1-1) was radioactively labeled using a Megaprimer-DNA labeling system (Amersham), and subjected to Southern hybridization.
  • Hybridization was performed according to a conventional method (Molecular cloning 2nd edn., Sambrook, J., et al., Cold Spring Harbor Laboratory U.S.A., 1989). As a result, it was thought that there were 4 genes in the Ec ⁇ 22I fragment of about 5.5 kb.
  • a library was prepared to clone the DNA fragment.
  • the chromosome of Candida od _ The DNA was cut with EcoT22I, and after agarose gel electrophoresis, a DNA fragment of about 5.5 kb was recovered from the gel. The recovered DNA fragment was ligated with pUC118 cut with PstI. After chilling, a library was prepared by transforming Escherichia coli DH5 ⁇ strain by the method of Hanahan (Gene, 10, 63 (1980)).
  • Plasmid pCPRA1 was cleaved with various restriction enzymes and analyzed by Southern hybridization. As a result, the PEP4 gene was found to be about 3.5 kb BglII-EcoT22 in Fig. 1. It was thought to be in region I. To determine the nucleotide sequence of this region, a 2.2 kb BglII-Ec0RV fragment (the region between Bg1II and EcoRV underlined in Figure 1) was blunt-ended. After the fragmentation, a 1.7 kb HindIII fragment (the region between the HindIIIs shown underlined in FIG.
  • the PEP4 gene was disrupted by transformation using the URA3 gene of Candi dabni dini as a marker.
  • a mutant strain of the URA3 gene of the Candidadabioidinicc ATCC 418800 strain CandidadaboidiniiSK6112 strain was used.
  • the Candidaboidinini SK612 strain was obtained according to a known method (Sakai Y. et al., J. Bacteriol., 173, 7458 (1991)).
  • a plasmid pDPRA1 in which about 2 kb of SnaBI—Ec0RV region of the gene was substituted with the URA3 gene was prepared. Reports of Sakai et al. In order to obtain Peracil-required strains again from gene-disrupted strains
  • the A3 gene having a repetitive structure was used as a marker before and after the structural gene.
  • the DNA fragment of 3.5 kb obtained by cutting p URP by Sa1I has about 0.9 kb of repetitive sequence before and after the URA3 structural gene (Fig. 2). .
  • pCPRA2 in which the P4 gene was inserted in the opposite direction to pCPRA1 was cut with SnaBI and EcoRV, and Xhol linker (Takara Shuzo) was inserted.
  • a 3.5 kb DNA fragment obtained by Sail cleavage of pURP was inserted into the Xh0I site of the obtained plasmid to obtain a plasmid pDPRA1 (FIG. 2).
  • the pDPRA1 obtained in (2_1) of this example was digested with Sa1I, and the Can didaboidinii SK612 strain was subjected to the lithium acetate method (Ito, H. et al., J. Bacteriol). , 153, 163 (1983)).
  • the obtained transformants were subjected to Southern analysis of their chromosomal DNA to screen for P.sodium 4 gene disrupted strains. That is, a 1.7 kb DNA fragment obtained by cutting the chromosome DNA of the host strain SK612 and the transformant with SalI and Ndel and cutting pCPRAl with SalI and SnaBI is obtained. Southern analysis was performed as a probe (Figure 3). As shown in Fig. 3, a band is detected at 3.8 kb in the host strain SK612, but a band is detected at 5.4 kb in the disrupted strain.
  • the disrupted strain was named C adcaboidinii SK 740 strain.
  • C andidaboidinii SK 740 strain After culturing C andidaboidinii SK 740 strain in a YPD medium until the stationary phase, a strain showing resistance to 5-fluorofluoridic acid (5-FOA) was obtained. Acquisition of 5-FOA resistant strains was carried out according to the method described in the experimental manual (Isao Ishida, Minoru Ando Z, Ed., Gene Expression Experiment Manual, Kodansha Scientific, 1994). A strain lacking the A3 gene was screened by performing Southern analysis in the same manner as when a gene-disrupted strain was obtained from the chromosomal DNA of the 5-F0A resistant strain. As shown in FIG.
  • Proteinase B gene of Candida do / d
  • PRB 5 5′—GGTAAYGGTCAYGGTACHCAYTGTGCH GGWAC-3 ′ (SEQ ID NO: 16);
  • PRB3 5 '-GCC ATWGAWGTAGCWGATAARACDGCW GTDGC-3' (SEQ ID NO: 17).
  • the primer P RB5 corresponds to the amino acid sequence GNGHGTHCAGT (SEQ ID NO: 14), and the primer PRB 3 is a sequence complementary to the base sequence corresponding to the amino acid sequence ATAVL SGT SMA (SEQ ID NO: 15).
  • the chromosomal DNA of the Candidaboidinii ATCC 48180 strain was mixed with the primers P RB5 and P RB3 and the PCR was performed using ExTaq polymerase (Takara Shuzo Co., Ltd. (30 seconds at 94 ° C, 50 ° C For 1 minute and 72 ° C for 2 minutes) X 30 cycles).
  • the amplified DNA fragment of about 0.5 kb was recovered and cloned into pT7BlueT-Vector (Novagen).
  • the chromosome DNA of Candida, boidini ATCC48180 strain was cut with various restriction enzymes, and subjected to 0.8% agarose gel electrophoresis. The separated DNA was transferred to HybondN + nylon membrane (Amersham).
  • the DNA fragment obtained in (3-1) of this example was radioactively labeled using a megaprimer DNA labeling system (Amersham), and subjected to Southern hybridization. As a result, it was shown that the PS1 gene was present in an EcoRI-HindIII fragment of about 5.5 kb and a BglII-EcoT22I fragment of about 4.5 kb. Next, about 5.5 kb of EcoR I—Hindii I I fragment, about 4.51?: 8 of 811 1— £ 13.
  • a library was prepared for cloning the fragment.
  • the chromosomal DNA of Candidabodidinii was cut with EcoRI and HindIII, and after agarose gel electrophoresis, a DNA fragment near 5.5 kb was recovered from the gel. The recovered DNA fragment was inserted between the Ec0RI and HindIII sites of pUC19 to prepare an EcoRI-HindIII plasmid rally.
  • a Bg1II-EcoT22I plasmid in which the Bg1II—EcoT22I fragment was inserted between the BamHI and PstI sites of pBluescript IISK + was prepared. did.
  • the transgene was disrupted by a transformation using the i? A3 gene of Candidaboidini as a primary candidate.
  • the Candidaboboidinii SK741 strain obtained in Example 2 was used.
  • Plasmid p DPRBl in which about 0.7 kb of the C1aI region of the PRB I gene was replaced with the A3 gene was prepared as follows.
  • pCPRBACla Approximately 2.0 kb DN obtained by digesting PC PRB 2 with Clal and EcoRI The A fragment was inserted into the CIaI-EcoRI region of pCPRBI. The resulting brassmid was named pCPRBACla.
  • pCPRBACla was cut with CIaI, blunt-ended with T4 DNA polymerase, and then Xhol linker was inserted.
  • a 3.5 kb DNA fragment obtained by cutting the p URP described in (2-1) of Example 2 by Sa1I was inserted into the Xh0I site of the obtained plasmid, and the plasmid p DP RB 1 was obtained ( Figure 5).
  • the pDPRBl obtained in (4-1) of this example was cut with HinclI and EcoRI, and the Candidnaboidinini SK741 strain was transformed by the lithium acetate method.
  • the resulting transformant was subjected to Southern analysis of the chromosome DNA, thereby screening a PRB1 gene-disrupted strain. That is, 1.3 kb obtained by cutting the chromosomal DNA of the host SK741 strain and the transformed strain with Bg1II and HindIII, and cutting pCPRB1 with C1aI and Bg1II. Southern analysis was performed using the DNA fragment as a probe (Fig. 6). As shown in Fig. 6, a band detected at 3 kb is detected in the host strain SK741, at 5.8 k in the disrupted strain.
  • the disrupted strain was designated as Candidabboidinii SK774 strain.
  • a 5-F0A-resistant strain was obtained from the CandidnabiodiniiSK7774 strain, and a strain lacking the URA3 gene was screened. Screening was performed by Southern analysis. As shown in FIG. 6, a band detected at the position of 5.8 k'b was detected in the SK774 strain, and was detected at a position of 3.2 kb in the strain lacking the URA3 gene.
  • the yeast was named Candidabboidiniini SK775 strain.
  • the protease activity of oidinii SK774 (pep4, prbl) strain and Ca ddaboidinii ATCC 48180 strain was measured. Each strain was cultured in a 2 ml YPD medium at 30 ° C until stationary phase.
  • the harvested cells were suspended in 0.2111 1 of 1001111 ⁇ Tris-HC1 buffer ( ⁇ 7.5), and 0.8 g of glass beads (0.425-0.6 mm, Sigma) ) was added, and the mixture was vigorously stirred for 1 minute and then cooled on ice for 1 minute, which was repeated 5 times.
  • the cell lysate was centrifuged at 10,000 rpm at 4 ° C for 10 minutes, and the supernatant fraction was obtained as a cell-free extract.
  • the protein concentration of the cell-free extract was measured using a protein assay kit (Bio-Rad).
  • the enzyme activity of the cell-free extract was determined by measuring the proteinase A activity and the carboxypeptidase Y activity according to the review of Jones (Jones, EW, Methods Enzymol., 194, 428 (1991)). That is, proteinase A activity was measured in a 1 ml reaction mixture containing 25 1 cell-free extracts, a final concentration of 100 mM Glycine-HC1 buffer (pH 3.2), and 1% acid-denatured hemoglobin. Measured at ° C. After 0 min, 10 min, 20 min, and 30 min, remove 200 t1 of the reaction solution, add 100 ⁇ l of 1 N perchloric acid, and rotate at 10,000 rpm for 10 min. Centrifuged.
  • Proteinase A activity was defined as the amount of enzyme that released 1 ⁇ g of peptide per minute per unit.
  • the ATCC 48180 strain detected 49.3 units of proteinase A activity per 1 mg of the cell-free extract, but the SK740 strain and the SK774 strain did not.
  • Carboxy peptidase Y activity 1 00 of the cell-free extract and 500 1 of mu 1 buffer (1 0 OmM T ris - HC 1 (p H 7. 5), 1 mM C a C 1 2) and 20 1 of A substrate solution (6 mM N-benzoyl-L-tyrosine-P-nitroanilide (Sigma) dissolved in dimethylformamide) was mixed, mixed well, and reacted at 37 ° C for 30 minutes. . The reaction was terminated by adding 600 1 of 1.5 ⁇ ⁇ acetic acid, filtered through a 0.22 ⁇ filter, and the absorbance at 405 nm was determined. Was measured.
  • Carboxypeptidase Y activity was defined as one unit of the enzyme which released 1 nmol of ⁇ -nitroaniline per minute. 0.72 units, 0.28 units, and 0.05 units of carboxypeptidase Y activity per mg of cell-free extract of ATCC 48180, SK740, and SK774, respectively was detected, and it was confirmed that carboxypeptidase Y activity was significantly reduced due to protease gene deficiency.
  • the previously reported human-derived cathepsin C gene was obtained by PCR, and the obtained DNA fragment was used as a probe. According to the nucleotide sequence of the human cathepsin C gene (Patris, et al., FEBS Lett., 369, 326 (1995)), the following oligonucleotides were synthesized:
  • HCat-3 5'-TCTGAGATTGCTGCTGAAAGTCTAC AGTCT-3 '(SEQ ID NO: 19).
  • QU I CK—Screen Huma nc DNA Library Panel (Clontech) was used as the rust-type DNA.
  • Type I DNA and primers HC at-5 and HC at-3 were mixed, and PCR was performed using Ex Taq polymerase (Takara Shuzo) (30 sec at 94 ° C, 30 sec at 60 ° C, 7 2 ° C for 2 minutes) X 3 0 cycle).
  • Ex Taq polymerase (Takara Shuzo) (30 sec at 94 ° C, 30 sec at 60 ° C, 7 2 ° C for 2 minutes) X 3 0 cycle).
  • a DNA fragment of about 1.2 kb amplified from a placenta-derived library was recovered and cloned into pT7BlueT-Vector (Novagen).
  • the nucleotide sequence was determined using Dye primer cycle sequencing FS Ready Reaction Kit (Nichikin Elma Co., Ltd.), and it was confirmed that the human-derived cathepsin C gene was inserted.
  • the inserted DNA fragment of 1.2 kb was prepared by digesting the plasmid with SmaI and XbaI, recovering it after agarose electrophoresis, and using it as a probe DNA fragment.
  • BB0 Vine Spleenc DNA library purchased from Stratagin, Inc. was used as a library for obtaining the cycatebucin C gene. Approximately 1,000,000 recombinant phageclones developed according to the attached protocol were subjected to risk cleaning with plaque hybridization. Infected with Escherichia coli XL1-Blue MR F 'strain together with the positive recombinant phage obtained and the helper phage attached to the library, and cultured at 37 ° C for 3 hours. The pB1uescript with was cut out.
  • the supernatant obtained by treating the culture solution at 70 ° C for 20 minutes was infected with Escherichia coli SOLR TM strain, and Escherichia coli having the recombinant plasmid DNA was selected by ampicillin resistance.
  • a 1 F 5′-GTACATATCCAGATCTATTAGGTACTTGG GTCTTTCAAGTTGGTTCTTCTGGTTCACAAAGAGAT GTTAATTGTTCTGTTATGGGTCCTCC AGAGAAGAAA
  • AIR 5'-GCAAACCATTTATAATCATTCAAGACAAT TTCGAAACCTT AGAAT T AC C AA CTTTAAGTGAACGACAACTTTCTTC- 3 '(SEQ ID NO: 21);
  • a 2 R 5′-CCCCCACTAGTCCTAGGACATCATGAACC C AACCTGTCATAGTTTC ATGAC AATAAGAAGT AACT TTACCACCTTCTTCTTTATATTTAAAGAAAGCAAAC CAT TTATAAT CAT TCAAGACAATTT CG—3 ′ (SEQ ID NO: 23);
  • B 1 F 5'-CGTTAATACTGCTAGATTAGCTGGTTTAG AAGAAACATACTCTAATAGATTATATCGTTATAATC ATGATTTCGTCAAAGCTATTAATGCTATTCAAAAAT CTTGGAC-3 '(SEQ ID NO: 24);
  • B 1 R 5 '-TACGAGAATGACCACCACCTCTTCTAATC ATTTCTTTAAGAGTTAATGTTTCATATTCCATATAA
  • B 2 F 5'-GGGGGGCGGCCGCGGGGCCTAGGTAGAAA TTGGGCTTGTTTCACTGGTAGAAAGACTGGTAATAC
  • AATCA—3 ′ (SEQ ID NO: 27);
  • C 1 F 5'-TTGCTTCTATGGGTATGATGGAAGCTAGA
  • C2F 5'-GGGGGACTAGTTGGGATTGGAGAAATGTT CATGGTATTAACTTTGTTACTCCTGTTAGAAATCAA GGTTCATGTGGTTCTTGTTACTCATTTGCTTCTATG GGTATGATGGAAGC TAGAATTAGAATTTTGAC-3 '(SEQ ID NO: 30);
  • C 2 R 5'—CC CC CAAGC TT CATTACAAC CAC CATAGA
  • D 1 F 5 '-AT TATAGAAAAGGTGTTTAT CAT CACAC T GGTTT AAT CATGCT
  • GCTGC TTCTG-3 ′ (SEQ ID NO: 32);
  • D 2 F 5 '-GGGGGAAGCTT TGATGAAATTAGAATTAG TTC ATCAAGGTCCTATGGC TGTTGCTTTTGAAGTCT
  • Region A was prepared by first mixing primers A1F and A1R and performing PCR using ExT : aq polymerase (Takara Shuzo) ((30 seconds at 94, 1 minute at 60, 72 minutes at 72). 30 seconds at 20 ° C.) ⁇ 20 cycles) to synthesize double-stranded DNA. After completion of the reaction, perform phenol black-mouthed form extraction and ethanol precipitation, and 1 the volume of the PCR reaction solution.
  • ExT aq polymerase
  • Regions B, C, and D were also synthesized using the primer shown in Fig. 7 in the same manner as in region A, and the plasmids pCT-B, pCT-C, p inserted into PB1uescript II KS +, respectively. CT-D was obtained.
  • Plasmid pECTC-S1 obtained in (6-2) of this example was digested with BamHI and transformed into C andidaboidinii SK612 and SK741 strains. Pick up 10 colonies of the resulting transformant for each host strain and place in the medium. The secreted cathepsin c activity was measured.
  • GLYS medium 1% 15.5 medium containing 3% glycerol, 0.67% yeast nitrase base and 0.5% yeast extract
  • the cells were collected by centrifugation at 3,000 rpm for 5 minutes.
  • the cells were harvested in the same amount of GLYS medium as MYS (methanol 5%, yeast nitrase base 0.67%, yeast extract 0). (55.5% medium containing 55.5%), and cultivated with shaking at 30 ° C for 48 hours.
  • the solution was concentrated 50-fold using 30 (Amicon), and the force-tepsin C activity was measured by the following method.
  • FIG. 9 shows the amount of cathepsin C produced by each transformant.
  • the use of the Candidaboidini J ′ strain in which the protease gene was disrupted as a host was superior in cathepsin C productivity.
  • SEQ ID NO: 9 Including a pre-region for proteinase A and a pro-maturation region for bovine cathepsin C, a Not I recognition site 5 'to the start codon (ATG) of the structural gene and 3' to the stop codon 1 shows the nucleotide sequence encoding the sequence.
  • SEQ ID NO: 12 shows a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 10.
  • SEQ ID NO: 13 Complementary to the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 11
  • SEQ ID NO: 16 shows the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 14.
  • SEQ ID NO: 17 Complementary to the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 15
  • SEQ ID NO: 20 shows primer A1F for PCR synthesis of DNA represented by SEQ ID NO: 9
  • SEQ ID NO: 21 shows primer A1R for PCR synthesis of DNA represented by SEQ ID NO: 9
  • SEQ ID NO: 22 Primer for PCR synthesis of DNA represented by SEQ ID NO: 9—A2F.
  • SEQ ID NO: 23 Shows a primer A2R for PCR synthesis of DNA represented by SEQ ID NO: 9.
  • SEQ ID NO: 24 shows primer 1B1F for PCR synthesis of DNA represented by SEQ ID NO: 9
  • SEQ ID NO: 25 shows primer B1R for PCR synthesis of DNA represented by SEQ ID NO: 9
  • SEQ ID NO: 26 Primer for PCR synthesis of DNA represented by SEQ ID NO: 9—B2F.
  • SEQ ID NO: 27 Shows a primer B2R for PCR synthesis of DNA represented by SEQ ID NO: 9.
  • SEQ ID NO: 29 Shows a primer C1R for PCR synthesis of DNA represented by SEQ ID NO: 9.
  • SEQ ID NO: 30 This shows a primer, C2F, for PCR synthesis of DNA represented by SEQ ID NO: 9.
  • SEQ ID NO: 31 shows primer C2R for PCR synthesis of DNA represented by SEQ ID NO: 9
  • SEQ ID NO: 32 Primer for PCR synthesis of DNA represented by SEQ ID NO: 9—D1F
  • SEQ ID NO: 33 shows a primer D1R for PCR synthesis of DNA represented by SEQ ID NO: 9
  • SEQ ID NO: 34 Primer for PCR synthesis of DNA represented by SEQ ID NO: 9—D2F
  • SEQ ID NO: 35 Shows a primer D2R for PCR synthesis of DNA represented by SEQ ID NO: 9.

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Abstract

On décrit des souches de Candida boidinii présentant une activité protéase réduite, et plus particulièrement des souches qui ont été inactivées au niveau de l'activité protéinase A et/ou protéinase B ; un procédé de production d'une protéine exogène utile (par exemple la cathepsine C) au moyen de la transformation d'une telle souche C.boidinii avec un vecteur d'expression contenant un gène codant la protéine exogène ; des protéines présentant l'activité protéinase A ou protéinase B ; des ADN codant ces protéines ; et un peptide signal sécrétoire de protéinase A provenant de $i(C.boidinii.)
PCT/JP1999/004802 1998-09-04 1999-09-03 Souches de candida boidinii presentant une activite protease reduite et utilisation de ces dernieres en tant qu'hote pour produire une proteine exogene WO2000014259A1 (fr)

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WO2003012098A1 (fr) * 2001-08-01 2003-02-13 Kirin Beer Kabushiki Kaisha Levure modifiee presentant un potentiel secretoire eleve de proteine etrangere et procede permettant de produire une proteine etrangere a l'aide de la levure
WO2003091431A1 (fr) * 2002-04-26 2003-11-06 Kirin Beer Kabushiki Kaisha Methylotrophe produisant une chaine de sucre de type mammifere

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JP4211603B2 (ja) 2001-05-29 2009-01-21 旭硝子株式会社 シゾサッカロマイセス・ポンベ（Ｓｃｈｉｚｏｓａｃｃｈａｒｏｍｙｃｅｓｐｏｍｂｅ）宿主の構築方法および異種タンパク質の製造方法
WO2016017693A1 (fr) * 2014-07-30 2016-02-04 第一三共株式会社 Procédé destiné à la production sécrétoire élevée améliorée de protéines

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003012098A1 (fr) * 2001-08-01 2003-02-13 Kirin Beer Kabushiki Kaisha Levure modifiee presentant un potentiel secretoire eleve de proteine etrangere et procede permettant de produire une proteine etrangere a l'aide de la levure
WO2003091431A1 (fr) * 2002-04-26 2003-11-06 Kirin Beer Kabushiki Kaisha Methylotrophe produisant une chaine de sucre de type mammifere
US7972809B2 (en) 2002-04-26 2011-07-05 National Institute Of Advanced Industrial Science & Technology Methylotrophic yeast producing mammalian type sugar chain

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