JPH08256770A - Protease derived from yeast belonging to the genus pichia - Google Patents

Abstract

PURPOSE: To obtain the subject protease useful for analyzing a C-terminal sequence of a protein, having substrate specificity. CONSTITUTION: This protease derived from yeast belonging to the genus Pichia has reactivity with an anti-baker's yeast carboxypeptidase Y antibody, and is extracted from a cultured supernatant liquid of Pichia pastoris GTS115, etc., as baker's yeast belonging to the genus Pichia and has the following physical and chemical properties. (1) Benzoyl-L-tyrosine-p-nitroanilide is hydrolyzed to liberate p-nitroanilide. (2) Protease activity; optimum pH is pH6 to 9. Deactivated by treatment at pH equal to or lower than about pH4 at about 60 deg.C for 1 hour. (3) Protease activity-optimal salt (NaCl) concentration is 0-1M. (4) Having four N-type saccharide chain bond sites and about 58kDa molecular weight (SDS-PAGE). (5) Having an amino acid sequence LysArgAsnAsnProGluValLeuLysVal in an N-terminal domain.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a protease derived from a yeast of the genus Pichia which is used as a host for an effective expression system for producing a recombinant product, a gene thereof and a method for producing the protease. The protease is involved in the degradation of the protein produced in the expression system using Pichia yeast as a host. Therefore, by elucidating the structure and the enzymatic function, it becomes possible to provide a method for preventing the decomposition of the protein product and improving the yield. Since the protease has a wide substrate specificity, it is highly likely to be widely used for C-terminal sequence analysis of proteins,
Development of a system for producing the enzyme in large quantities in Pichia yeast is extremely useful.

The present invention further provides a DNA obtained by modifying the gene for a protease derived from the yeast of the genus Pichia and the DN.
The present invention relates to a modified Pichia yeast strain having A, and a method for producing a heterologous protein using the modified Pichia yeast strain as a host. According to the protein expression system using the modified Pichia yeast strain as a host, the protein can be produced in good yield.

The present invention also relates to a pre-peptide (signal peptide) useful for secretory expression of a heterologous protein in a yeast of the genus Pichia, and a secretory expression system for the heterologous protein and a secretory expression system utilizing the pre-peptide. The present invention relates to a method for producing a heterologous protein used.

[0004]

[Prior Art] [Problems to be Solved by the Invention] Pichia pastoris which is a methanol-assimilating yeast
Etc.) have recently attracted attention as effective hosts for heterologous protein expression systems. Alcohol oxidase, which is located at the first position in the methanol assimilation pathway, is an enzyme that is very strongly inducibly expressed in the presence of methanol, and many results have been found in expression systems in which the promoter is connected upstream of the target protein gene. (Bio / technology 11, 905-909). However, when a heterologous protein is produced by a gene recombination method, the problem is not limited to the expression system of the yeast of the genus Pichia, and always poses a problem that the target product is degraded by the protease derived from the host.

In the process of investigating the expression system of a heterologous protein using Pichia yeast, the present inventors (i) that a chymotrypsin-like protease is released in the culture supernatant of Pichia yeast, (ii) The protease activity is suppressed by a serine protease inhibitor, (iii) Degradation of the target heterologous protein product proceeds in the cells of the host (Pichia yeast) and in the culture supernatant thereof, etc. I got the knowledge of. Therefore, if the properties of such a protease derived from the yeast of the genus Pichia can be clarified and the expression of the protease can be controlled at the gene level, it is very effective for the development of a heterologous protein expression system using the yeast of the genus Pichia as a host. It is believed that there is. However, at present, little research has been conducted on such proteases relating to Pichia yeast.

Therefore, it is an object of the present invention to provide a protease derived from a yeast of the genus Pichia, a DNA having a nucleotide sequence encoding the protease, and a method for producing the protease. The present invention also comprises a DNA modified so that the production of a functional product of the protease gene is at least suppressed, and the presence of the DNA suppresses the protease-producing ability as compared with a native Pichia yeast. It is an object of the present invention to provide a modified Pichia yeast strain and a method for producing a heterologous protein characterized by using the modified Pichia yeast strain as a host. A further object of the present invention is to provide a prepeptide derived from the protease of the present invention as a signal peptide useful for a Pichia yeast secretory expression system, and to provide a method for producing a heterologous protein using the prepeptide.

[0007]

Under the circumstances described above, the present inventors have been conducting research to elucidate the properties of the enzyme released into the culture supernatant of the yeast of the genus Pichia. Succeeded in isolating a polypeptide having a chymotrypsin-like activity from Kiyomizu, and confirmed that the enzyme is involved in the degradation of a protein product in an expression system using a yeast of the genus Pichia as a host, thus completing the present invention. did.

That is, the present invention relates to the following protease derived from the yeast of the genus Pichia. (1) A protease derived from a yeast of the genus Pichia having reactivity with an anti-baker's yeast carboxypeptidase Y antibody. (2) The Pichia yeast-derived protease according to (1), which has the following characteristics. (i) At least phenylmethylsulfonyl fluoride (hereinafter referred to as PMSF) inhibits the protease activity. (ii) The synthetic substrate benzoyl-L-tyrosine-p-nitroanilide (Bz-Tyr-pNA) is decomposed to release p-nitroanilide (pNA). (iii) having a molecular weight of about 58 kDa (sodium dodecyl sulfate-polyacrylamide gel electrophoresis, hereinafter SD
S-PAGE) (iv) It has four N-type sugar chain binding sites. (V) Protease activity optimum pH is 6-9. (vi) It shows no activity when treated at pH 4 or lower, or at 60 ° C. for 1 hour. (vii) Protease activity optimum salt (NaCl) concentration is 0-
1M. (3) The Pichia yeast-derived protease according to (1) or (2), which has substantially the following amino acid sequence in the N-terminal region.

[0009]

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(4) The Pichia yeast-derived protease according to any one of (1) to (3), which has substantially the following amino acid sequence.

[0011]

[Chemical 9]

(5) The precursor before being processed by a protease in a host cell has a prepeptide region, a propeptide region and a protease region, and the prepeptide region has the following amino acid sequence: The protease derived from yeast of the genus Pichia according to any one of (1) to (4).

[0013]

[Chemical 10]

(6) The precursor before being processed by a protease in a host cell has a prepeptide region, a propeptide region and a protease region, and the propeptide region has the following amino acid sequence: The protease derived from the yeast of the genus Pichia according to any one of (1) to (5), characterized in that

[0015]

[Chemical 11]

Further, the present invention is a DNA containing a base sequence encoding the above-mentioned Pichia yeast-derived protease, preferably a DNA having the following base sequence:
And a method for producing the above-mentioned Pichia yeast-derived protease.

[0017]

[Chemical 12]

The present invention also relates to a DNA obtained by modifying a part of a DNA containing a nucleotide sequence encoding a protease derived from Pichia yeast so that the production of a functional product of the DNA is at least suppressed, preferably a DNA. Regarding a DNA in which a transformation marker gene is inserted into a coding sequence of a DNA containing a nucleotide sequence encoding the protease, and further by having the modified DNA, the protease activity is suppressed as compared with a natural Pichia yeast strain. And a modified Pichia enzyme strain.

Furthermore, the present invention provides a prepeptide contained in a precursor of a protease derived from a yeast of the genus Pichia, a DNA encoding the peptide, a vector for secretory expression of a heterologous protein and a secretory expression of a heterologous protein characterized by having the DNA. The present invention relates to a method for producing a heterologous protein, which comprises culturing a yeast of the genus Pichia having the heterologous protein secretory expression vector, and collecting the heterologous protein from the culture supernatant.

Hereinafter, the present invention will be described in detail. (1) Pichia yeast-derived protease The protease of the present invention is a protein (peptide) degrading enzyme that is originally produced by Pichia yeast, and is characterized by having reactivity with an anti-baker's yeast carboxypeptidase Y antibody. To do.

The yeast of the genus Pichia from which the protease of the present invention is derived is not particularly limited, but specifically, Pichia
pastoris, Pichia finlandica, Pichia trehalophila,
Pichia koclamae, Pichia membranaefaciens, Pichia o
puntiae, Pichia thermotolerans, Pishia salictaria,
Pichia guercuum Pichia pijperi etc. are illustrated.

In the present invention, "anti-baker's yeast carboxypeptidase Y antibody" means baker's yeast, preferably Saccharomyces cerevisiae.
The antibody having the property of binding to carboxypeptidase Y (EC3.4.16) produced by Escherichia coli. This can be conveniently obtained by a conventional method such as the antiserum obtained by administering carboxypeptidase Y to a mammal such as rabbit as an immunogen. The term “reactive” means that the protease is recognized as an antigen by the anti-baker's yeast carboxypeptidase Y antibody and binds by the antigen-antibody reaction.

The protease of the present invention preferably further has the following features. (i) The activity is inhibited by at least PMSF. PMSF
Is a compound that does not inhibit the activity of metalloproteases, most thiol proteases and acid proteases, but inhibits the activity of serine proteases, commonly referred to as serine protease inhibitors. In the present invention, “inhibition” is a concept including not only the case of completely eliminating the protease activity but also the case of diminishing the original activity.

(Ii) The synthetic substrate Bz-Tyr-pNA is decomposed to release pNA. The synthetic substrate is generally used as a substrate for measuring the activity of chymotrypsin or carboxypeptidase Y [Method in Enzymology].
185, 372 (1990)]. From this property, the protease of the present invention is presumed to be a protease having a substrate specificity and reaction mechanism similar to chymotrypsin or carboxypeptidase Y.

(Iii) SDS-PAGE shows a molecular weight of about 58 kDa. In addition, SDS-PA in the present invention
The molecular weight can be determined by GE by the method of Laemmli (Nature, 2
27, 680, 1976). In particular,
The protease of the present invention was heat-treated at 100 ° C. for 3 minutes in the presence of SDS (0.1%) and 2-mercaptoethanol (1%), and then about 1 mm in the presence of SDS (0.1%).
Electrophoresis using a 4-20% polyacrylamide slab gel, the location of migrated protease is detected by staining, and it is determined by comparison with a standard protein of known molecular weight.

(Iv) It has four N-type sugar chain binding sites.
Generally, in an N-type sugar chain-binding protein, Asn-X-Thr or Asn-X-Ser in its amino acid sequence is used.
It is known that sugar is added to Asn (X means any amino acid residue). From this, the protease of the present invention has Asn-X-T in its amino acid sequence.
owns 4 hrs or Asn-X-Ser, N
It is thought that it possesses four types of sugar chain binding sites.

(V) Peptidase activity The optimum pH is 6-9,
The pH is preferably 7 to 9, and more preferably 7 to 8. Specifically, the protease of the present invention has at least a pH of 6 to 9 when using a buffer solution having various pHs according to the protease activity measurement method shown in Reference Example 1.
It preferably exhibits high protease activity in the range of pH 7-9, more preferably pH 7-8.

(Vi) It shows no activity when treated at a pH of about 4 or lower, or at 60 ° C. for 1 hour. Specifically, the protease of the present invention at least exhibits activity below the detection limit at pH 4 or less and at pH 10 or more when using buffer solutions having various pHs according to the method for measuring protease activity shown in Reference Example 1. It has properties. Furthermore,
The protease of the present invention has the property of losing its activity when incubated at various temperatures according to the method for measuring protease activity shown in Reference Example 1 after incubation for at least 60 ° C. for 1 hour.

(Vii) The optimum activity salt (NaCl) concentration is 0 to
It is 1M, preferably 0 to 0.75M. In particular,
The protease of the present invention has at least various salt concentrations (NaC) according to the method for measuring protease activity shown in Reference Example 1.
In the case of using the buffer solution having 1), the salt (NaCl) concentration is slightly reduced to 1 M, but 0 to 0.75 M
It has the property of exhibiting almost the same activity within the range.

The protease of the present invention is not particularly limited as long as it is derived from a yeast of the genus Pichia and has the above-mentioned characteristics, but the N-terminal region is preferably SEQ ID NO.
Is a protease having an amino acid sequence represented by,
More preferably, it is a protease having substantially the amino acid sequence shown by SEQ ID NO: 2. In addition, such an amino acid sequence may be partially modified (for example, substitution, deletion or insertion of amino acid residues or peptide chains) within a range that does not change the above-mentioned characteristics.

Further, the protease of the present invention is expressed and produced as an inactive precursor form (prepro form) in yeast of the genus Pichia, and the prepeptide region and propeptide region are sequentially cleaved in the processing process to obtain a mature protease. Is an enzyme. That is, in the protease of the present invention, the precursor before being processed by the mature protease in yeast cells of the genus Pichia has a prepeptide region, a propeptide region and a protease region.

The pre-peptide region is a peptide fragment that generally serves as a signal for a protein to pass through the endoplasmic reticulum membrane, and is cleaved at a stage after the protein passes through the membrane and enters the endoplasmic reticulum. . Examples of the peptide fragment preferably include those having the amino acid sequence shown by SEQ ID NO: 3.

The propeptide region is a peptide fragment generally contained in an inactive precursor of a protein, and the peptide fragment is cleaved and removed from the inactive precursor by a specific protease to give an activity specific to the protein. Is expressed. Examples of the peptide fragment preferably include those having the amino acid sequence represented by SEQ ID NO: 4.

The mature protease means a protease finally produced, that is, the protease of the present invention having the above-mentioned characteristics. Further, the precursor of the protease of the present invention is more preferably exemplified by one having the amino acid sequence shown in SEQ ID NO: 5.

The protease derived from the yeast of the genus Pichia of the present invention is similar to baker's yeast, preferably carboxypeptidase Y derived from Saccharomyces cerevisiae in enzymatic properties and immunological reaction, and the protease of the present invention. The amino acid sequence represented by SEQ ID NO: 2 exemplified as the mature form of Bacillus subtilis has a high homology with the amino acid sequence of the mature form of baker's yeast carboxypeptidase Y. Therefore, carboxypeptidase Y derived from Pichia yeast Is likely to be.

The protease of the present invention is obtained by culturing a yeast of the genus Pichia according to a conventional method, preferably under conditions suitable for the growth of the yeast, and extracting and purifying from the culture supernatant or cultured cells by a conventional method. It can be manufactured. Culture in a high-density culture system is preferable. This is because by such culture, a part of the protease produced by the yeast of the genus Pichia is released to the outside of the yeast and can be easily isolated from the culture supernatant. Further, the protease of the present invention can also be produced by a method commonly used by those skilled in the art, such as recombinant DNA technology or polypeptide synthesis method.

When using recombinant DNA technology, the protease of the present invention is a D encoding the amino acid sequence.
NA, preferably a host cell transformed with an expression vector containing a DNA encoding the amino acid sequence of the precursor of the protease of the present invention is cultivated in a medium, and the desired protease is collected from the culture. You can In this case, as the host cell, a commonly used microorganism [bacteria (for example, Escherichia coli etc.), yeast (for example,
Saccharomyces cerevisiae, Pichia pastoris, etc.), animal cells, etc.], but preferably yeast, especially Pi
chia pastoris. The expression vector is not particularly limited as long as it functions so as to express and produce the protease of the present invention, and can be appropriately selected depending on the host cell used. Suitably, alcohol oxidase 1 (AOX), which is strongly transcribed in the host cell and is preferably inducible.
1), mutant alcohol oxidase 2 (mAOX2)
(Ohi, H et al., Mol.Gen.Genet., 243, 489-499, 1994
D) encoding the amino acid sequence of the precursor of the protease of the present invention downstream of the promoter fragment of a gene such as
An expression vector containing an expression unit in which DNAs of NA and 3'untranslated region are connected together with a gene for a transformation marker such as SUC2, HIS4, URA3, and G418 resistance gene. According to the expression vector, a transformant By culturing, the protease of the present invention can be produced in a large amount. Further, as a transformation method, a culturing method (conditions such as medium, temperature and time), a method usually used in this field can be used. The following DNA may be mentioned as a DNA encoding the amino acid sequence of the precursor of the protease of the present invention.

(2) DNA Containing Nucleotide Sequence Encoding Pichia Yeast-Derived Protease The DNA of the present invention is characterized in that it contains the above-mentioned Pichia yeast-derived protease-derived nucleotide sequence of the present invention. is there. The base sequence is not particularly limited as long as it is a base sequence capable of encoding the protease derived from the yeast of the genus Pichia of the present invention, but preferably the base sequence encoding the amino acid sequence shown in SEQ ID NO: 5 (amino acid numbers 108 to 522), More preferably, the base sequence shown in SEQ ID NO: 5 (base numbers 2386 to 3633) can be mentioned. DNA of the present invention
May have any of the above-mentioned base sequences, for example, a DNA having a base sequence encoding the propeptide region of the protease of the present invention at the 5'end side of the base sequence, and further having a protease of the present invention at the 5'end side thereof. Examples thereof include DNA having a nucleotide sequence encoding the pre-peptide region.
As such DNA, specifically, a DNA characterized by the restriction enzyme map shown in FIG. 5, more specifically, amino acid numbers 21 to 523 or amino acid numbers 1 to 523 in the amino acid sequence shown in SEQ ID NO: 5. DNA encoding the sequence shown by, more preferably SEQ ID NO: 5
The DNA having the base numbers 2125 to 3633 or the base numbers 2065 to 3633 in the base sequence shown in is exemplified.

Among the DNAs of the present invention, the DNA encoding the precursor (prepro-form) of the protease of the yeast of the genus Pichia of the present invention is the gene P encoding the carboxypeptidase Y of baker's yeast, especially Saccharomyces cerevisiae.
It has some similarities with the RC1 gene. Therefore, hereinafter, for convenience, this gene will be referred to as the Pichia PRC1 gene (P-
(PRC1 gene). However, P of the present invention
-Nucleotide sequence of PRC1 gene and PRC1 derived from baker's yeast
It is essentially different from the base sequence of a gene (hereinafter, also referred to as S-PRC1 gene).

The DNA containing the nucleotide sequence encoding the protease derived from the yeast of the genus Pichia of the present invention can be produced by a conventionally known method. For example, based on the nucleotide sequences exemplified in the present invention, a DNA synthesizer is used to synthesize a part or all of the DNA, or a yeast of the genus Pichia (for example, P.
It can also be produced by amplification using a chromosomal DNA of P. pastoris) by the PCR method.

The DNA containing a nucleotide sequence encoding the protease derived from the yeast of the genus Pichia of the present invention is provided for the first time by the present invention as a gene of carboxypeptidase Y produced by the yeast of the genus Pichia. Therefore, the present invention is extremely useful for elucidating the processing process of carboxypeptidase Y derived from Pichia yeast in a Pichia yeast cell.

Further, the protease of the present invention acts to lower the molecular weight of a product produced by using yeast of the genus Pichia as a host cell, especially the protease sensitive protein, and loses the function of the product. Therefore, the elucidation of the protease gene according to the present invention is a method of reducing or eliminating the protein-lowering activity of the protease at the gene level in order to favorably operate the expression system of the recombinant product using Pichia yeast as a host. It also leads to provision. That is, the P-PRC1 gene of the present invention (hereinafter, referred to as modified P-PRC1 described below will be used to reduce or eliminate the protein depolymerization activity of the Pichia yeast-derived protease of the present invention.
In order to distinguish it from a gene, a native P-PRC1 gene) can be achieved by modifying the gene so that it cannot produce a functional product.

(3) DNA obtained by modifying the natural P-PRC1 gene Accordingly, the present invention provides a modified product of the natural P-PRC1 gene,
That is, a part of the base sequence of the natural P-PRC1 gene (DNA containing a base sequence encoding a protease derived from the yeast of the genus Pichia) is modified so as to at least suppress the production of a functional product of the gene (DNA). DNA
Regarding

Here, "functional product of P-PRC1 gene"
The term "protease produced by the natural P-PRC1 gene", that is, the protease of the present invention means
As long as it has the same function as the protease, it is included in the functional product here. Here, the "function" means the protease activity of the protease derived from the yeast of the genus Pichia of the present invention, specifically, at least the "substrate Bz-Ty".
The activity of “degrading r-pNA and releasing pNA” is meant. The term "at least suppress the production of functional products" means not only the case where the product is not expressed and the product encoded by the P-PRC1 gene is not produced at all, but the product obtained by the expression is P-PRC1.
A product which is not the same as the functional product of the PRC1 gene and whose function is attenuated (that is, the product has no protease activity of the functional product of the native P-PRC1 gene and the function of the native P-PRC1 gene) When the protease activity is lower than the protease activity of the product).

Therefore, the mode of modification of the gene may be one that disables the expression of the gene or modified P-PR.
The expression / product of the C1 gene is not particularly limited as long as it does not have the protease activity originally possessed by the product of the natural P-PRC1 gene or is attenuated as compared with it. Specifically, natural P-PRC
Illustrative is a modification in which at least one nucleotide in the nucleotide sequence of one gene is deleted or at least one nucleotide is inserted in the sequence. Due to such modification, the reading frame is displaced, and the function of the product that is not expressed or that is obtained even when expressed is different from the function of the product derived from the natural gene.

A preferred modification method is natural P-PR
A method of inserting a transformation marker gene into the C1 gene coding region can be mentioned. According to this, the natural type P
-There is an advantage that the PRC1 gene can be disrupted and that a mutant having the modified P-PRC1 gene can be screened using the introduced transformation marker gene as an index.

Examples of the transformation marker gene to be used include P. pastoris or S. cerevisiae HIS4 gene, ARG4 gene, URA3 gene, SUC2 gene, G418 resistance gene and the like. Preferably,
It is the SUC2 gene.

(4) Modified Pichia yeast strain The modified Pichia yeast strain of the present invention is the above-mentioned modified P-PRC.
It is a yeast strain belonging to the genus Pichia in which the protease-producing ability is suppressed as compared with the yeast strain belonging to the genus Pichia based on having one gene. That is, it is a yeast of the genus Pichia having the above-mentioned modified P-PRC1 gene in place of the natural P-PRC1 gene, and does not produce a functional product of the natural P-PRC1 gene at all, To produce products with diminished protease activity.

Such a modified Pichia yeast strain can be prepared by various methods. For example, modification of the natural P-PRC1 gene in the natural Pichia yeast or random mutation of the natural Pichia yeast strain is carried out to suppress the protease activity as compared with the natural Pichia yeast strain. The method of selecting the mutants can be mentioned. The method for producing a modified Pichia yeast strain by modifying the natural P-PRC1 gene is specifically described as natural P-PRC1.
It is carried out by introducing a modified gene at a specific locus of the gene by a site-specific integration method. The modified gene is integrated by replacing the endogenous gene of the host. A convenient method for introducing the modified gene into the target locus of the yeast host is to include the modified gene in a linear DNA fragment with two homologous ends of the two native genes in the host. Is. This directs the transformation so that its expression product undergoes homologous recombination at specific sites in the gene that affect protease activity.

A preferred method is a method for transforming a native Pichia yeast using the modified P-PRC1 gene of the present invention. As a method for transforming the natural yeast of the genus Pichia and a method for culturing the yeast cell, an ordinary method adopted in the art can be used. For example, as a transformation method, a spheroplast method [Creggh et al., Mol.
ell.Biol., 5,3376 (1985), U.S. Pat.No. 4,879,231.
No.], lithium chloride method [Ito et al., Agric.Biol.Che
m., 48,341 (1984), European Patent Application No. 312,934, and US Patent No. 4,929,535].

The host cell derived from a natural yeast of the genus Pichia used for transformation is not particularly limited, but is preferably a methanol-utilizing yeast (methylotrophi) that can efficiently utilize methanol as the sole carbon source and energy source.
c) Yeast. As a suitable methanol-assimilating yeast, specifically, auxotrophic Pichia pastoris GTS1
15 (NRRL Y-15851), Pichia pastoris GS190 (NRRLY-18014), Pichia pastoris PPF1 (NRRL Y-18017), wild type Pichia pastoris strain (NRRL Y-11430,
NRRL Y-11431) and the like.

Further, more preferably, a strain in which at least one autotrophic marker gene is deleted, for example, a HIS4 deficient Pichia strain, GS115 (ATCC208).
64), ARG4-deficient Pichia strain, GS190. HIS4
/ URA3-deficient Pichia strain, GS4-2. HIS4 / AR
G4-deleted Pichia strain PPF1 (NRRL Y-1801
7: US Pat. No. 4.812.405) and the like. Thus, when the host cell is a strain lacking at least one autotrophic marker gene, the modified P-PRC1
It is preferable to use a gene having an autotrophic marker gene that is deleted in the host cell. According to such a method, a transformant (modified Pichia yeast strain) into which a modified P-PRC1 gene has been incorporated can be identified quickly and easily, and it is useful.

The modified Pichia yeast strain further comprises a natural medium [eg, YPD medium (1% yeast extract, 2% peptone, 2% glucose), YPM medium (1
% Yeast extract, 2% peptone, 2% methanol, etc.] and the like, and retains the same growth ability as the natural Pichia yeast strain. This means that the presence or absence of modification of the P-PRC1 gene does not affect the growth of Pichia yeast under nutritional conditions. Therefore, the modified Pichia yeast strains of the present invention are excellent hosts for the production of protease-sensitive products from Pichia yeast. That is, since the yeast has a decreased or eliminated protease activity derived from the host cell as compared with the natural Pichia yeast strain, a protease-sensitive heterologous protein can be efficiently produced.

The heterologous protein is not particularly limited as long as it is sensitive to a protease derived from a yeast of the genus Pichia, and specifically, epidermal growth factor (EG
F), growth hormone releasing factor (GRF), insulin-like growth factor-1 (IGF-1), IGF1 binding protein 3 (IGF1BP3), human serum albumin (HS
A), pro-urokinase-annexin V fusion protein,
Examples include chymase and the like.

Expression systems useful for the production of heterologous proteins are:
It can be produced by various methods. For example, in the modified Pichia yeast strain described above, D encoding a heterologous protein is added.
Method for introducing NA, natural P-PRC possessed by recombinant Pichia yeast strain having DNA encoding heterologous protein
Method of mutating one gene subsequently to the embodiment of the modified P-PRC1 gene of the present invention, or method of simultaneously transforming a native Pichia yeast strain with the above-mentioned modified P-PRC1 gene and a DNA encoding a heterologous protein Etc.

The recombinant protein expression system Pichia yeast has at least a promoter region, a DNA encoding a desired heterologous protein, and a transcription terminator region in the direction of the reading frame of transcription. These DNAs are DNAs encoding the desired heterologous protein
As transcribed into NA, they are arranged in functional relation to each other.

As a promoter, P. pastoris AO
X1 promoter (promoter for the primary alcohol oxidase gene), P. pastoris AOX
2 promoters (promoters for the secondary alcohol oxidase gene), P. pastoris DAS
Promoter (promoter for dihydroxyacetone synthase gene), P. pastoris P40 promoter (promoter for P40 gene), P. past
The oris aldehyde dehydrogenase gene H can also have a pH suitable for the growth of host cells and the production of a desired protein. Further, aeration and stirring can be added if necessary.

After culturing, the culture supernatant is recovered, and the desired heterologous protein can be purified and obtained from the supernatant by a method known per se, such as fractionation method, ion exchange, gel filtration or affinity column chromatography.

[0059]

INDUSTRIAL APPLICABILITY The protease of the present invention is similar to carboxypeptidase Y of baker's yeast in terms of its immunological properties, enzymatic reaction properties, amino acid sequence and the like. Therefore, the carboxypeptidase derived from Pichia yeast is Inferred to be Y. Therefore, the present invention for the first time provides the structure of carboxypeptidase Y derived from yeast of the genus Pichia, the enzymatic function, the structure of a precursor involved in processing, and the gene of the enzyme. Moreover, since the protease of the present invention has broad substrate specificity, it is highly likely to be widely used for C-terminal sequence analysis of proteins and the like. Therefore, the method for producing a protease of the present invention is extremely useful in that the enzyme can be obtained in a large amount.

Further, elucidation of the enzyme and its gene can provide a method for preventing the decomposition of the enzyme-sensitive protein product in the expression system using Pichia yeast as a host and improving the yield of the protein. That is, the modified Pichia yeast strain of the present invention has a growth ability equivalent to that of the natural Pichia yeast strain, and has reduced or eliminated protease activity as compared with the natural Pichia yeast strain. Therefore,
According to the expression system using the modified Pichia yeast strain of the present invention as a host, a protease-sensitive heterologous protein can be efficiently produced.

The present invention also provides a pre-peptide which is a precursor of a protease derived from Pichia yeast as a useful secretion signal in a heterologous protein expression system using Pichia yeast as a host. Such a prepeptide processes a target heterologous protein in an accurate position in an expression system using Pichia yeast as a host. Therefore, by using the pre-peptide, the target heterologous protein can be secreted and produced, and the production of a heterologous protein having a function and structure closer to that of a natural protein can be achieved as compared with a system that accumulates in cells. Can be expected. Further, in a system in which the target protein accumulates in cells, it is necessary to destroy the cells in order to purify the target protein and purify the target protein from the disrupted solution, but according to the present invention, such purification is required. The protein can be isolated easily without any treatment. That is, the present invention provides efficient expression of a heterologous protein using Pichia yeast as a host.
It also provides a new expression / production system useful for production.

The plasmids, enzymes such as restriction enzymes, T4 DNA ligase and other substances used in the examples of the present invention are commercially available and can be used according to a conventional method. Those skilled in the art are also familiar with the procedures used for cloning cDNA, determining the nucleotide sequence, transforming host cells, culturing transformed cells, collecting enzymes from the resulting culture, and purifying. There is something that can be known from the literature.

[0063]

EXAMPLES AND EXPERIMENTAL EXAMPLES Hereinafter, the present invention will be described in more detail based on Examples, Experimental Examples and Reference Examples. However, the present invention is not limited to this.

Example 1 Isolation and purification of protease from culture supernatant of Pichia yeast (1) Pichia yeast (strain name: Pichia pastoris GTS1)
15) using a jar fan mentor to obtain a cell density of about 8
High-density culture was performed until it reached × 10 ⁹ cells / ml, and the obtained culture supernatant (about 600 ml) was concentrated 10 times. The concentrated solution was dialyzed with 20 mM Tris-HCl buffer (pH 7.0) containing 20 mM CaCl ₂ and then equilibrated with the same Q sepharose FF anion exchange column (φ1.
6 × 25 cm, manufactured by Pharmacia LKB Biotechnology Co., Ltd.). After washing the column with the same buffer,
Was eluted by the linear gradient method using the same buffer solution containing 0.3 M NaCl, and the effective fraction having protease activity was collected. Tween 2 is added to 110 ml of this fraction.
0 was added to a final concentration of 0.05%, and the mixture was concentrated to 8 ml. The concentrated solution was added to 20 mM CaCl ₂ , 0.3M
20 mM Tris-HCl buffer containing NaCl (p
H7.0) TSKgel G3000SW equilibrated with H7.0)
Gel filtration was performed using a column (φ2.15 × 60 cm, manufactured by Tosoh Corporation). Effective fraction having protease activity 48.
After concentrating 5 ml 3 times, the solvent of the concentrate is 0.5M.
50 mM Tris-HCl buffer containing NaCl (p
H7.0) and was added to a copper chelate column (φ2.15 × 60 cm, Pharmacia LKB Biotechnology) equilibrated with the buffer solution. After washing the column with the same buffer, elution was carried out by a linear gradient method using the same buffer containing 0 M to 0.03 M of imidazole to collect 12.6 ml of an effective fraction having protease activity, Fraction solvent containing 20 mM CaCl ₂ 2
The medium was replaced with 0 mM Tris-HCl buffer (pH 7.0). DEAE-Sepharose FF equilibrated with the same buffer
It was added to an anion exchange column (φ0.5 × 20 cm, manufactured by Pharmacia LKB Biotechnology) and eluted by a linear gradient method using the same buffer solution containing 0 M to 0.3 M NaCl, and 9 m having protease activity.
1 effective fraction was collected. In addition, Tween2
0 was added to a final concentration of 0.02%, and the mixture was concentrated to 2 ml. The concentrated solution was added to 20 mM CaCl ₂ ,
TSKgel G300 equilibrated with 20 mM Tris-HCl buffer (pH 7.0) containing 0.3 M NaCl
Gel filtration was carried out again on a 0SW column (φ2.15 × 60 cm, manufactured by Tosoh Corporation) to obtain 3.6 having protease activity.
5 ml of the effective fraction was used as a purified protease solution. still,
The protease activity of each eluted fraction was measured by the method described in Reference Example 1 below.

Table 1 shows the purification efficiency of protease in each purification step. By these purification operations, the specific activity of protease increased about 162 times and the recovery rate was 1.32.
%Met.

[0066]

[Table 1]

(2) Next, the obtained purified protease solution was subjected to reduction treatment in the presence of 0.1% SDS, 1% 2-mercaptoethanol, and sodium dodecyl sulfate-poly (polyamide) was used using a 4-20% polyamide gradient gel. Acrylamide gel electrophoresis (SDS-PAGE) was performed, and a protein band was detected by silver staining (manufactured by Wako Pure Chemical Industries, Ltd.). As a result, the purified protease has a molecular weight of about 58.
It was detected as a single band in the kDa portion. From this, it was confirmed that the protein having protease activity, which was purified from the culture supernatant of Pichia yeast, was a single-chain polypeptide having a molecular weight of about 58 kDa (FIG. 1).

(3) The proteins separated by SDS-PAGE were transferred to a PVDF membrane and subjected to Western blotting with a rabbit anti-S. Serevisiae CPY antibody. Rabbit anti-S. Serevisia
As the e-CPY antibody, an antiserum obtained by immunizing rabbits with carboxypeptidase Y (manufactured by Sigma) derived from baker's yeast (S. cerevisiae) was used. This was treated with a goat anti-rabbit antibody horseradish peroxidase conjugate (manufactured by Bio-Rad) and developed with 4-chloro-naphthol. As a result, a band having a molecular weight of about 58 kDa was detected as a colored band, and the protease derived from the yeast of the genus Pichia isolated in (1) above was used as a rabbit anti-S. Serevisia.
It was confirmed to have reactivity with the eCPY antibody (Fig. 2).

(4) Further transferred PVDF
The region around 55 to 60 kDa of the membrane was cut out and subjected to N-terminal amino acid analysis by the Edman method, and the N-terminal amino acid sequence was determined up to 30 residues. As a result, S.serevis
N-terminal amino acid sequence of carboxypeptidase Y, a serine protease of iae (Valls et al., Cell 48, 8
87-897, 1987) and showed very high homology (Fig. 3).
From the above results, it was strongly suggested that the protein having protease activity isolated and purified from the culture supernatant of Pichia yeast was carboxypeptidase Y derived from Pichia yeast.

Example 2 Acquisition of DNA having a nucleotide sequence encoding a protease derived from Pichia yeast (DNA encoding a precursor) N-terminal amino acid sequence of a protease derived from Pichia yeast and baker's yeast (S. cerevisiae) Based on the finding in Example 1 that it has high homology with that of mature carboxypeptidase Y of Bacillus subtilis, the PRC1 gene encoding carboxypeptidase Y of baker's yeast (hereinafter referred to as S-PR
C1 gene) was amplified by the PCR method to be used as a probe, and the chromosomal gene of Pichia yeast was subjected to Southern analysis to clone a gene encoding a protease derived from Pichia yeast.

(1) Southern hybridization of chromosomal gene of Pichia yeast using S-PRC1 gene (i) Amplification of S-PRC1 gene (PCR method) S-PRC1 gene of 2,632 bp which has been reported ( Valls et al., Cell, 48, 887-897, 1987) by PCR
It was amplified by the method. More specifically, first, the S-PR
Positive strand primer a (5′-GGAATTCA) in which EcoRI sites were added to complementary sequences at both ends of C1 gene.
TCGATTTCCGTATATAGGATG-3 ') and the primer b (5'-GGAATTCCAGCT) on the reverse chain side.
GATTTTGTTAGTTATG-3 ′) was chemically synthesized. Using these primers, the conventional method (Sherman et a
l., Yeast DNA isolation. In: Laboratory course manu
al for methods in yeast genetics, Cold Spring Harb
or Laboratry, 1986).
PCR was performed using the genomic DNA of AH22 strain as a template. The amplified 2.6 kb DNA band was excised, purified and subcloned into the EcoRI site of pUC19 to prepare pHO104 (FIG. 4).

(Ii) On the other hand, yeast of the genus Pichia (P. pastoris)
The chromosomal DNA derived from the GTS115 strain is routinely used (Sherman et al.
al., Yeast DNA isolation. In: Laboratory course m
anualfor methods in yeast genetics, Cold Spring Ha
rbor Laboratry, 1986), digested with various restriction enzymes (EcoRI, Xbal, BamHI, BglII), and subjected to agarose gel electrophoresis to transfer the isolated DNA to a nylon membrane.
The S-PRC1 gene cloned by the PCR method was transformed into Ba
After digestion with mHI, a 1.1 kb fragment corresponding to the translation region was prepared as a probe, and the fragment was prepared according to a conventional method (Sambrook et al., Mole.
cular cloning: A laboratory manual. Cold Spring H
Arbor Laboratory, 1989) (hybridization at 55 ° C, washing at 42 ° C with 2 x SSC-
0.1% SDS, and 0.2xSSC-0.1% SD
S), and Southern hybridization was performed. As a result, it was predicted that the gene encoding the protease of the Pichia genus yeast (hereinafter referred to as P-PRC1 gene) has the restriction enzyme map shown in FIG.

(2) Cloning of P-PRC1 gene (see FIG. 6)
It was carried out by cloning the 5'untranslated region of the RC1 gene and the 5'side region of the translated region and the 3'side region and 3'untranslated region of the translated region of the P-PRC1 gene. (i) Cloning of 5'-untranslated region of P-PRC1 gene and 5'-side region of translated region The chromosomal DNA of Pichia yeast GTS115 strain was EcoR
Digested with I, the digested DNA separated on an agarose gel,
A region corresponding to a size of 3 to 3.5 kb was separated and purified. The separated DNA was λExCell EcoRI /
Ligation was performed with CIP (Pharmacia). Add the ligation mixture to Gigapack Gold in vitro p
It was inserted into a phage using an ackaging kit (manufactured by Stratagene), and Pichia DNA / EcoRI / 3-3.5k
b / λExCell library was constructed (pHO1
05, FIG. 6).

The host strain E. coli obtained by culturing the λExCell library overnight. E. coli NM502 infected with NZY
Agarose plate (1% NZ amine, 0.5% N
aCl, 0.5% yeast extract, 0.2%
MgSO ₂ .7H ₂ O, 1.5% agar) 3
Plaque formation was performed by culturing at 7 ° C. for 16 hours. The plaque was then transferred to a nylon membrane. Using a 1.1 kb BamHI fragment corresponding to the translation region of the S-PRC1 gene as a probe, the conventional method (Sambrook et al., Molecular cloning: A laborator
plaque hybridization was performed according to the Cold Spring Harbor Laboratory, 1989) (55 ° C).
Hybridization and washing at 2 ℃ at 42 ℃
SSC-0.1% SDS, and 0.2xSSC-0.
Performed by 1% SDS). E. coli capable of releasing a phagemid with a positive insert. coliNP
66 is 2 × YT (0.8% polypeptone, 0.5% yeast extract, 0.5% NaCl) -50 μg
/ Ml spectinomycin, 30 μg / ml chloramphenicol, 0.2% maltose in culture medium at 32 ° C
It was cultured for 4 hours. Collect the cells and add 3 ml of NZCVM to the cells.
The cells were suspended in (NZY + 0.1% casamino acid, 50 μg / ml spectinomycin). The bacterial suspension was left at 39 ° C for 20 minutes and mixed with positive plaques.
The infection was established by standing for 0 minute. Then 2 x YT-50
μg / ml spectinomycin was added, and the mixture was cultured with shaking at 32 ° C. for 1 hour. L containing 100 μg / ml ampicillin
-A small amount of the above culture solution was added to broth, and the mixture was cultured at 37 ° C overnight, and then a phagemid was prepared from the grown bacterial cells. The prepared phagemid was E. It was amplified by retransformation of E. coli DH5.

(Ii) In vitro PCR cloning of the 3'side region and 3'untranslated region of the P-PRC1 gene translation region (Isegawa et al., Molecular and Cellular
(See Probes 6, 467-475, 1992) OUT primer having a sequence homologous to the 3'th position to the 3123rd position (refer to base number 5 in the sequence listing) from the 5'end of the 3.2 kb EcoRI fragment cloned by the above method:
5'-AGATGGGGGACCCTATCAGAGA-
IN primer having a sequence homologous to 3'and 3124 to 3143: 5'-ACTGGGAAGAATG
TGTATGA-3 'was synthesized. PUC18 was used as the cloning vector. The chromosomal DNA of P. pastoris GTS115 strain was digested with Xbal-BglII. The digested DNA was separated on an agarose gel, and D at around 1.3 kb was separated.
The NA region was separated and purified. The separated DNA and the Sau3AI cassette were ligated, purified, and then used for 1 s using C1 primer (Takara Shuzo) and OUT primer.
t PCR was performed. Next, 2nd PCR was performed using a part of the reaction solution of 1st PCR as a template and C2 primer (Takara Shuzo) and IN primer. The reaction solution was subjected to agarose gel electrophoresis, and an amplification band around 0.6 to 0.7 kb was cut out and purified. The amplified fragment is BamHI
-Digested with BglII and inserted into the BamHI site of pUC18 to create pHO109 (see Figure 6).

The pHO105 obtained in (i) was added to XhoI-
3.2 kb fragment digested with BamHI and isolated, pH
The 3.4 kb fragment isolated by digesting O109 with SalI-BamHI was ligated to P-PRC1.
A plasmid pHO110 was constructed in which the entire region of about 3.8 kb from the 5'untranslated region of the gene to the 3'untranslated region via the translation region was inserted (Fig. 6).

(3) Determination of nucleotide sequence of P-PRC1 gene The entire nucleotide sequence of the P-PRC1 gene cloned in pHO110 was determined from both directions by the primer walking method. That is, M13-40 primer (manufactured by Pharmaci LKB Biotechnology) and dideoxy method (Sanger et al., Proc Natl Acad Sci USA
74, 5463-5467, 1977). After that, design a primer at an appropriate position, perform primer walking,
Nucleotide sequences were sequentially determined using DNASIS (Hitachi Software Engineering). In the sequence listing SEQ ID NO: 5,
1 shows the entire nucleotide sequence of the P-PRC1 gene and the amino acid sequence of a Pichia yeast-derived protease deduced therefrom. Further, FIG. 7 shows the homology of the amino acid sequences of the protease derived from the yeast of the genus Pichia and S-CPY.

The open reading frame of P-PRC1 gene had a total length of 1,569 bp. There is no typical TATAbox (TATAAA) in the 5'untranslated region and the similar sequence "TATAGA" is ATG upstream-51.
It was found at position (SEQ ID NO: 5, base numbers 2014-2019).
The Pichia yeast-derived protease deduced from the open reading frame consists of 523 amino acids, and the N-terminal amino acid sequence of the protease purified from the culture supernatant and the deduced Pichia yeast-derived protease amino acid position 108 (SEQ ID NO: The sequence from lysine (see 5) was completely matched. Therefore, the gene encoding the protease derived from the yeast of the genus Pichia was cloned into P-
It was confirmed to be the PRC1 gene. Signal sequence breakpoint prediction (von Heijne, Nucleic. Acids. Res. 14, 4
683-4690, 1986), the prepeptide was 20 amino acids from methionine to glycine at amino acid position 20 (see SEQ ID NO: 5). Further, it was suggested that the mature protease of the present invention is a polypeptide chain from the amino acid position 108 (SEQ ID NO: 5) by analogy with the N-terminal amino acid of the purified protease.
The protease has a pre-sequence of 20 amino acids, a pro-sequence of 87 amino acids, and a mature sequence of 416 amino acids. The molecular weights of the polypeptide chains without sugar chains are 59.44 kD for the precursor and 47.35 kD for the mature protein. Was calculated. In addition, a sequence consisting of Asn-X-Thr or Asn-X-Ser was found in 4 positions in the amino acid sequence (the position of Asn, in the base number 5 in the sequence listing, amino acid positions 193, 271, 484). And 487
Position), it was considered that there are four N-type sugar chain binding sites. Serine, which is presumed to be the active center, is S-CP.
It was presumed to be serine at the 249th amino acid position (SEQ ID NO: 5), as estimated from the homology with Y. The amino acid homology with S-CPY was 61%. Although the homology near the active center of the mature protein showed a high level,
The pre-peptide region and the vicinity of the C-terminal region had low homology.

Example 3 Preparation of P-PRC1 gene disruption strain S. cerevisiae SUC2 gene functions as a transformation marker for P. pastoris (Sreekrishna et al., Ge
ne 59, 115-125, 1987) was used for gene disruption. That is, the NsiI-SphI region of about 0.3 kb containing the active center site of the translation region of the P-PRC1 gene was
Plasmid p that has been replaced with S. cerevisiae SUC2 gene
HO115 (Fig. 8) was prepared and PstI of pHO115 was prepared.
The -AccIII fragment was introduced into the host strain GTS115. Specifically, the plasmid pHO110 in which the full-length P-PRC1 gene was cloned was digested with HindIII and self-ligated to construct pHO114. From pMM022 in which the S. cerevisiae SUC2 gene has been cloned, PstI-SphI was used for SU.
The C2 gene was excised, and NsiI-Sp of pHO114 was excised.
It was inserted into the hI site to construct P-PRC1 gene substitution disruption type plasmid pHO115 (FIGS. 9 and 10). The resulting pHO115 was cleaved with PstI-AccIII to isolate a fragment containing the 5.3 kb SUC2 gene, and
storis GTS115 strain with lithium acetate method (Ito et al.,
J. Bacteriol. 153, 163-168, 1983).
Msu plate (amino acid-free 0.67% YN
B, 0.5% sucrose, 1.5% agar) was used. Sucrose-assimilating colonies on the Msu plate were picked up, and single colony isolation was performed again on the Msu plate to isolate the sucrose-assimilating strain. Southern analysis was performed on several isolated strains to screen for P-PRC1 gene-disrupted strains. That is, the sucrose assimilation strain and the host GTS115 strain were cultured in 2 ml YPD medium (1% yeast extract, 2% peptone, 2% glucose) at 29.5 ° C. and 170 rpm for 3 days, and the method of Sherman et al. (Sherman et al., Y
east DNA isolation. In: Laboratory course manual f
or methods inyeast genetics, Cold Spring Harbor La
Chromosomal DNA was prepared according to Boratry, 1986). The obtained chromosomal DNA was digested with EcoRI and subjected to 1% agarose gel electrophoresis.

0.9 kb EcoRI-PstI fragment of the 5'untranslated region of the P-PRC1 gene, 0.5 kb NsiI-SphI fragment of the coding region, and SU
A 1.2 kb BamHI fragment on the 3'side of the C2 gene was prepared as a probe fragment, and Southern hybridization was performed by a conventional method (Sambrook et al., Molecular cloni.
ng: A laboratory manual. Cold Spring Harbor Labor
atory, 1989). The results are shown in FIGS.
As shown in Fig. 13 and Fig. 13, the bands detected in the host GTS115 strain are 3.2 kb and 3.2 kb, respectively, and there is no detection band, while the bands detected in the sucrose assimilating strain are 5.8 kb and the detection band, respectively. None, 5.8k
It was b. Therefore, the sucrose assimilation strain is PP
It became clear that the RC1 gene was disrupted.

The disrupted strain was named P. pastoris DIPY115 strain. Disrupted strain DIPY115 strain and host GTS115
The strain was cultivated in 50 ml of YPD medium and the total OD ₅₄₀ value was 3
3 ml of intracellular fraction was prepared from the equivalent of 000 bacterial cells, and
When z-Tyr-pNA degrading activity was measured, GTS
The 115 strain had an activity of 7 μg / ml, but the DIPY115 strain had an activity of 1 μg / ml (FIG. 14). Therefore, it was concluded that the expression of the Pichia yeast-derived protease was suppressed by the disruption of the P-PRC1 gene. FIG. 15 shows the results of comparing the cell growth rates in this culture. As for the cell growth rate, the initial cell amount was OD ₅₄₀ =
50 ml YPD and YPM medium to 0.1
It was planted in a flask and cultured at about 30 ° C. and 140 rpm. It was determined by collecting a small amount of the culture solution every 24 hours and measuring the OD ₅₄₀ value. As a result, the growth of the DIPY115 strain was equivalent to that of the host strain regardless of whether the carbon source was glucose or methanol.

Example 4 (1) Preparation of P-PRC1 gene overexpressing strain 3 base number 2062 of start codon of P-PRC1 gene
Has a sequence homologous to position 2984 (SEQ ID NO: 5),
PCR primer (5'-CCAGGCCTCAATGATATTTACA with StuI site added to the 5'end
CACCTATAT-3 ') and a PCR primer (5'-CCAGGCCTGATC) having a sequence homologous to the reverse chain of nucleotide positions 3850 to 3829 (SEQ ID NO: 5) of the 3'untranslated region and having a SutI site added to the 5'end.
PCR was performed using TTCCCGTTGTACGADATA-3 ') and a plasmid pHO110 in which the full-length P-PRC1 gene was cloned as a template plasmid to amplify the open reading frame and 3'untranslated region of the P-PRC1 gene. The resulting PCR product was digested with SutI to obtain plasmid pAO80.
0.9 obtained from 7N (JP-A-2-104290).
A plasmid pHO that connects to the AsuII site at the 3'end of the AOX1 promoter of kb and inserts this and S. cerevisiae SUC2 gene into pUC19 to overexpress the P-PRC1 gene under the control of the AOX1 promoter.
118 was constructed (Figure 16).

Next, the S. cerevisiae SUC2 gene was transformed into P.
Functions as a transformation marker for storis (Sreekrishn
a et al, 1987) was used to produce overexpressed Pichia yeast. That is, pHO118 is changed to ClaI
It was cut into a linear form by cutting with, and introduced into the P. pastoris GTS115 strain by the lithium acetate method (FIG. 17). In addition, Ms was used as the regeneration medium for the transformant that acquired the sucrose assimilation ability.
u-plate (0.67% amino acid free YNB, 0.
5% sucrose, 1.5% agar) was used.
Sucrose-assimilating colonies on the Msu plate were picked up with a toothpick, and single colony isolation was performed again on the Msu plate to isolate the sucrose-assimilating strain. Southern analysis was performed on several isolated strains to obtain pHO1.
18 was integrated at the P-PRC1 locus PP
The RC1 gene overexpressing strain was screened. That is,
2 ml of sucrose utilization strain and host GTS115 strain
YPD medium (1% yeast extract, 2% peptone, 2% glucose) at 29.5 ° C., 170 rp
The cells were cultured for 3 days, and the cells were analyzed by the method of Sherman et al.
Chromosomal DNA was prepared according to Herman et al., 1986). Chromosomal DNA was digested with PstI-BglII,
1% agarose gel electrophoresis was performed. 0.6 kb BglII-NsiI fragment of AOX1 promoter (probe 1) and 1.6 kb PstI- of P-PRC1 gene.
An XbaI fragment (probe 2) was prepared as a probe fragment, and Southern hybridization was carried out by a conventional method (Sambrook e
al., 1989). The results obtained by using the probe 1 are shown in FIG. 18, and the results obtained by using the probe 2 are shown in FIG. From the Pichia transformant that acquired the sucrose assimilation ability, pHO118 was converted into P-PRC1.
As a result of Southern hybridization screening of the P-PRC1 gene overexpressing strain integrated in the locus, U8-21 strain was obtained.

Example 5 (1) Preparation of secretory expression vector using pre-peptide sequence of Pichia yeast-derived protease as secretory signal sequence Encodes pre-peptide signal sequence of Pichia yeast-derived protease shown in SEQ ID NO: 3 in Sequence Listing The human serum albumin (HS
A) The DNA shown in FIG. 20 was synthesized in order to ligate to the N-terminus of the DNA encoding the mature sequence of the gene. The synthesized DNA contains the pre-sequence of the P-PRC1 gene and natural H
It is directly linked to the mature sequence of the SA gene. This D
AsuII and DraI sites were added at both ends in order to exchange the NA fragment with the pre- and pro-sequences of the native HSA gene. Anneal this pieces to each other and
After cutting with uII-DraI, HSA secretion expression plasmid p
MM042 [JP-A-4-29984, Japanese Patent Application No. 6-
291323 (paragraph numbers 0018 to 0020), FIG.
0]] and obtained by cutting with DraI-PstI.
It was ligated with a fragment of mature HSA of kb. The obtained DNA fragment was ligated with the vector fragment obtained by cutting pMM042 with AsuII-PstI to construct pKM148.

PKM148 is a plasmid in which mature HSA is secreted by the signal sequence of P-PRC1 under the control of the mutant AOX2 promoter. Also pK
By digesting M148 with the restriction enzyme StuI,
It is possible to transform the host by cutting it at a single site in the HIS4 gene, which is a selectable marker, into a linear form.

(2) P. pastoris GT by expression vector
Transformation of S115 strain (1) 10 μg of pKM148 DNA obtained in (iii)
And 10 μg of control pMM042 DNA were added to S
After digestion with tuI, the P. pastoris GTS115 (his4) strain was treated with the lithium chloride method (Ito et al., Agric. Biol. Chem., 48,3).
41, 1984, etc.). The resulting transformant was inoculated into 50 ml YP + 2% methanol broth,
Culturing was carried out at 30 ° C. for 3 days. After completion of the culture, HSA secreted in the supernatant was confirmed by SDS-PAGE.

(3) N-terminal amino acid sequence of secreted HSA The N-terminal amino acid sequence of secreted HSA was determined. As a result, as shown in Table 2, pMM042 and pKM148
The correct amino acid sequence of mature HSA was detected in each of the derived transformants, and it was confirmed that the N-terminal was normally processed.

[0088]

[Table 2]

Experimental Example 1 Properties of Pichia yeast-derived protease (1) Inhibition of protease activity by protease inhibitor (phenylmethylsulfonyl fluoride: PMSF) -1 Purified Pichia yeast protease obtained in Example 1
PMSF was added to 100 μl of a solution of (3,200 μg eq./A280) diluted 50 times with distilled water so that the final concentration was 1 mM, 10 mM or 20 mM, and the mixture was allowed to stand at room temperature for 2 hours. The protease activity in these treatment solutions was measured by Bz-Tyr-p.
The degradation activity of NA was measured as an index. The results are shown in Fig. 21. As a result, the activity of protease was completely inhibited by 10 mM or more of PMSF. From this, it was suggested that this protease is a serine protease.

(2) Inhibition of protease activity by PMSF-2 P8-PRC1 gene overexpression U8-obtained in Example 4
Twenty-one strains were planted in 50 ml of YPM medium using a flask and cultured by shaking at 29.5 ° C. and 140 rpm for 48 hours (OD ₅₄₀ = 300), and intracellular fractions were prepared by the glass bead method. PMSF was added to 100 μl of this fraction at a final concentration of 1 m.
M, 10 mM or 20 mM was added thereto, and the mixture was allowed to stand at room temperature for 2 hours. The protease activity in these treatment solutions was measured using the decomposition activity of Bz-Tyr-pNA as an index.
As a result, protease activity was completely inhibited when PMSF was 10 mM or more (FIG. 22). From this, PP
It was suggested that the protease produced in large quantities from the RC8 gene overexpressing U8-21 strain is a serine protease.

Experimental Example 2 Protease properties derived from yeast of the genus Pichia-optimum pH of protease-As shown in Table 3, Bz-Tyr- adjusted to various pH values.
Buffer for pNA degrading activity (0.1M Tris-HC
Bz-Tyr-p of the protease derived from the yeast of the genus Pichia prepared in Example 1 using 1, 1 mM CaCl ₂ )
The NA degrading activity was measured. As a result, it was revealed that the optimum pH of the protease is 5 to 9, preferably about 7 to 9.

[0092]

[Table 3]

Experimental Example 3 Protease characteristics derived from yeast of the genus Pichia-optimum salt concentration of protease-Bz-Tyr-pNA degrading activity buffer (0.1M T
NaCl was added to ris-HCl, 1 mM CaCl ₂ ) to adjust various salt concentrations shown in Table 4. Using these buffers, B of the purified protease prepared in Example 1
The z-Tyr-pNA decomposition activity was measured. As a result, there was no particular difference in the activity of the protease within the NaCl concentration range of 0 to 0.75M, and the activity was about 64% at 1M.
The activity was reduced to (Table 4).

[0094]

[Table 4]

Experimental Example 4 Protease Properties from Pichia yeast-Temperature Stability of Protease-The purified protease prepared in Example 1 was used at 30 ° C or 6 ° C.
After incubating at 0 ° C for an arbitrary time, the activity of the protease was measured using the degradation of Bz-Tyr-pNA as an index. As a result, the activity remained at 48 ° C. for 48 hours at 30 ° C., whereas it was completely deactivated at 60 ° C. for 1 hour (Table 5).

[0096]

[Table 5]

Experimental Example 5 Protease derived from U8-21 strain Natural Pichia yeast (P. pastoris GTS115) strain, P-PRC1 gene disrupted strain DIPY1 prepared in Example 3
The protease activity of each of the culture supernatants of the 15 strains and the P8-PRC1 gene overexpressing U8-21 strain prepared in Example 4 and the intracellular fraction was measured and compared. Specifically, each strain was planted in a 50 ml YPM medium (1% Bacto yeast extract, 2% Bacto peptone, 2% methanol) / flask so that the initial bacterial cell amount would be OD ₅₄₀ value = 0.1, and 29. It was cultured at 5 ° C. and 140 rpm for 72 hours. After culturing, centrifugation (2,000 xg, 5 minutes) was performed to separate into a supernatant and cells, and the supernatant was used as a culture supernatant. On the other hand, for the bacterial cells, 15 ml of Tris-HCl (pH 7.5)
Wash twice with 3 ml Tris-HCl, pH 7.5.
And 1 ml of glass beads were added and crushed by vigorous stirring. The supernatant clarified by repeating centrifugation was used as the intracellular fraction liquid (3 ml).

As a result, GTS11 was used as the culture supernatant.
No protease was detected in 5 strains and DIPY115 strain, and 4 μg / ml protease was detected only in U8-21 strain (FIG. 23, A). Intracellular fraction (3 ml)
For DIPY115 strain, only very weak protease activity was detected, but about 7 μg / ml protease was detected in GTS115 strain, and P
-In the PRC1 gene overexpression strain U8-21 strain, 2000 μg, which is about 300 times that of the natural GTS115 strain
/ Ml or more protease was detected (Fig. 23,
B).

Reference Example 1 Measurement of Protease Activity Using synthetic Bz-Tyr-pNA (manufactured by Nacalai Tesque, Inc.) as a substrate, the protease activity was measured using this decomposition activity as an index. Specifically, 100 μl of sample, 500 μl of buffer solution [0.1 M Tris-HCl (pH 7.0), 1 mM CaCl ₂ ] and 2 were added to a polypropylene test tube.
After adding 0 μl of a substrate solution (30 mg / ml Bz-Tyr-pNA solution prepared by dissolving in dimethylformamide) and stirring well, the mixture was incubated at 37 ° C. for 1 hour. The reaction was stopped by adding 600 μl of 1.5 M acetic acid thereto, and the mixture was clarified by filtration with 0.22 μm Milex GV (manufactured by Millipore). Using this as a measurement sample, 40
The absorbance ( _A405 value) at 5 nm was measured. In addition,
Carboxypeptidase Y derived from chymotrypsin (manufactured by Sigma) or baker's yeast (S. cerevisiae) as a standard product
(Manufactured by Sigma) was used. That is, 2 of the standard enzyme
0, 10, 5, 2.5, and 1.25 μg / ml solutions were prepared, and using this as a sample, a standard curve of A ₄₀₅ value was prepared, and the protease activity of the measurement sample was calculated from this.

[0100]

[Sequence list]

SEQ ID NO: 1 Sequence length: 10 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Origin: P. pastoris

SEQ ID NO: 2 Sequence Length: 416 Sequence Type: Amino Acid Topology: Linear Sequence Type: Peptide Origin: P. pastoris Sequence Lys Arg Asn Asn Pro Glu Val Leu Lys Val Asp Phe Thr Lys Gln 1 5 10 15 Tyr Ser Gly Tyr Leu Asp Val Glu Ala Asp Asp Lys His Phe Phe 20 25 30 Tyr Trp Phe Phe Glu Ser Arg Asn Asp Pro Gln Asn Asp Pro Ile 35 40 45 Ile Leu Trp Leu Asn Gly Gly Pro Gly Cys Ser Ser Leu Thr Gly 50 55 60 Leu Phe Phe Glu Leu Gly Ser Ser Arg Ile Asn Glu Asn Leu Lys 65 70 75 Pro Ile Phe Asn Pro Tyr Ser Trp Asn Gly Asn Ala Ser Ile Ile 80 85 90 Tyr Leu Asp Gln Pro Val Asn Val Gly Phe Ser Tyr Ser Ser Ser 95 100 105 Ser Val Ser Asn Thr Val Val Ala Gly Glu Asp Val Tyr Ala Phe 110 115 120 Leu Gln Leu Phe Phe Gln His Phe Pro Glu Tyr Gln Thr Asn Asp 125 130 135 Phe His Ile Ala Gly Glu Ser Tyr Ala Gly His Tyr Ile Pro Val 140 145 150 Phe Ala Asp Glu Ile Leu Ser Gln Lys Asn Arg Asn Phe Asn Leu 155 160 165 Thr Ser Val Leu Ile Gly Asn Gly Leu Thr Asp Pro Leu Thr Gln 170 175 180 Tyr Arg Tyr Tyr Glu Pro Met Ala Cys Gly Glu Gly Gly Ala Pro 185 190 195 Ser Val Leu Pro Ala Asp Glu Cys Glu Asn Met Leu Val Thr Gln 200 205 210 Asp Lys Cys Leu Ser Leu Ile Gln Ala Cys Tyr Asp Ser Gln Ser 215 220 225 Ala Phe Thr Cys Ala Pro Ala Ala Ile Tyr Cys Asn Asn Ala Gln 230 235 240 Met Gly Pro Tyr Gln Arg Thr Gly Lys Asn Val Tyr Asp Ile Arg 245 250 255 Lys Glu Cys Asp Gly Gly Ser Leu Cys Tyr Lys Asp Leu Glu Phe 260 265 270 Ile Asp Thr Tyr Leu Asn Gln Lys Phe Val Gln Asp Ala Leu Gly 275 280 285 Ala Glu Val Asp Thr Tyr Glu Ser Cys Asn Phe Glu Ile Asn Arg 290 295 300 Asn Phe Leu Phe Ala Gly Asp Trp Met Lys Pro Tyr His Glu His 305 310 315 Val Ser Ser Leu Leu Asn Lys Gly Leu Pro Val Leu Ile Tyr Ala 320 325 330 Gly Asp Lys Asp Phe Ile Cys Asn Trp Leu Gly Asn Arg Ala Trp 335 340 345 Thr Asp Val Leu Pro Trp Val Asp Ala Asp Gly Phe Glu Lys Ala 350 355 360 Glu Val Gln Asp Trp Leu Val Asn Gly Arg Lys Ala Gly Glu Phe 365 370 375 Lys Asn Tyr Ser Asn Phe Thr Tyr Leu Arg Val Tyr Asp Ala Gly 380 385 390 His Met Ala Pro Tyr Asp Gln Pro Glu Asn Ser His Glu Met Val 395 400 405 Asn Arg Trp Ile Ser Gly Asp Phe Ser Phe His 410 415

SEQ ID NO: 3 Sequence Length: 20 Sequence Type: Amino Acid Topology: Linear Sequence Type: Peptide Origin: P. pastoris Sequence Met Ile Leu His Thr Tyr Ile Ile Leu Ser Leu Leu Thr Ile Phe 1 5 10 15 Pro Lys Ala Ile Gly 20

SEQ ID NO: 4 Sequence Length: 87 Sequence Type: Amino Acid Topology: Linear Sequence Type: Peptide Origin: P. pastoris Sequence Leu Ser Leu Gln Met Pro Met Ala Leu Glu Ala Ser Tyr Ala Ser 1 5 10 15 Leu Val Glu Lys Ala Thr Leu Ala Val Gly Gln Glu Ile Asp Ala 20 25 30 Ile Gln Lys Gly Ile Gln Gln Gly Trp Leu Glu Val Glu Thr Arg 35 40 45 Phe Pro Thr Ile Val Ser Gln Leu Ser Tyr Ser Thr Gly Pro Lys 50 55 60 Phe Ala Ile Lys Lys Lys Asp Ala Thr Phe Trp Asp Phe Tyr Val 65 70 75 Glu Ser Gln Glu Leu Pro Asn Tyr Arg Leu Arg Val 80 85

SEQ ID NO: 5 Sequence Length: 3850 Sequence Type: Nucleic Acid Number of Strands: Double Strand Topology: Linear Sequence Type: Genomic DNA Origin: Organism Name: P. pastoris Strain Name: GTS115 Sequence Feature Symbol representing the feature: sig peptide Location: 2065..2124 Method by which the feature was determined: p Feature symbol: Location: 2125..2385 Method by which the feature was determined: p Symbol representing the feature: mat peptide Location : 2386..3633 method to determine the characteristics: p sequence GAATTCACCT GCTCAATGAC TTTACTTAAG TGAGTGTAAG CCTCTTGTGG AGACAATTTC 60 AGTTCGGTGA TTAAACGATC AACTTTGTTC AATACAAGAA TAGCTTTCAA CTTGTCTGAC 120 CAAACTTGTC GTAGCACACT AATTGTCTGA GAACAAACTC CTTCCACGAC GTCGACTAAT 180 ACAACTGCCC CATCACATAA TCTCGAAGCT GTACTAACTT CTGAGCTAAA GTCAACATGA 240 CCTGGAGAAT CAATTAAATT AATAAGATGT TCATTGACTG TGATATCACT ATCAGAATCA 300 CCCTCATTTC TGTGTATCAC CCTAAAATAC AGCGAAACAG CGCTACTCTC CATTGTTATG 360 CCCCGTAACT GCTCATCCTC CCTTGAGTCC AAGTATCTAA TCTTGCCCGC CATTTTGTG T 420 GAGATTATGC CATTTGTCGC TAAGAGACTA TCGCTTAAAG AAGTCTTACC ATGGTCCACA 480 TGAGCAACTA TACATATATT TCGAACACAA TCGGGGTCAT TTTGAAGAGT GTTCAGTTGT 540 TCTTTAGTTA ACCGCATAGT TACCAAAACT CGGTAACGAA CAAATGGTAT CACGCTCCTC 600 CAATAACTAG TAAAAAAAAA AACTAATCCG CGCACAAGCT TAAATCGATT ATATAATGAT 660 ATACCACCCA GGGGCAATTA ACCTTTGAAA ATATAATGAT TTCTATCCAA TTGCACTACT 720 AGGCTCTCGG TTATCTTTTT CATCAAAAAA TCCGTTTGTC CAGGGTACAG CTCCATCTGT 780 GTTAGAGACC GAAAATTCCA AGTCTTCAGT TGTAATCATG TCCCAATACT TGTACAGCAT 840 CATCACTGCG AATAGATTGC AAAGTGTACC AAAAAACAGT CCATCAAGGT AATGATTAAC 900 ACCCTCGCAA ATCTGATTTG AAAAACGATA CATGAGAACC TGTGCTGCAG TGAAAAAGAA 960 ACATCCTAAC ATGATTGCAC CCAAAGCCCA CCAGTTTTTC AAAATAGTCA GTATAAGAAT 1020 GATTTGAGAG AGTACGTAAA TGCCAATAGT GACCGCATTG AGGAAGTACA TGGTCACGAA 1080 AAGAGCGAGT GTATTTGTGT TATCGACAAT TGAGTTACTG GACCAATTGT GAAAAGTGAA 1140 AAGCGAAAGG ATAAAGTTAA GGGAAAAGGC ACATAAAGCA ATCAATCTAG TAAAGTTAAT 1200 AGATTTTCTT GTGCCATCTT CCCAAAACTG AAACCCCAGA AAACCATTCA GCATCAGAGA 1260 CCAGCAGCA G GCTGATGCCA GGCCAATTTG AACTGCCACG AAGTATCCGT ATGTTGCACT 1320 GCTGGGTGGA GAAACACCAG CATCAACGAC CAATGAGGCG ATGGTGTTGG CAATGTACAG 1380 GTAGAAAAAA AAAAGCATTT CGATCCTTCC AATTGCTGTG TATTTGCTTC TAACATGGAA 1440 CACAATAATC ATCAAGACGA CAATAGCGCT GATGTGTATA AAACAGTTCC CTACCTGGAA 1500 AATCATTGTG TTGGTTACCG ATATACTTCT AGCGTAACAA GTAGAAATAA TTCCCTTGAA 1560 GTTTGTACCG TGGTTGGGCA GTAAAACATT ACACAGAGGT AATGGAGTTT TTTGACAAAT 1620 CTCATAAAAG TTCCCAAATG ACATAACTTG TTTCAGTTAA TGAGCTACTT CTATGCCTTC 1680 GTTATAACTT TATATATGGT TGTCAAATTC CTCGCCTGAA TCTGAAACCA AGATATATTA 1740 TATACTTATG TAAGCATCCT TACCTCTGTC CTGCTTCACT TTTTATGTAC CTCAGCCAAT 1800 AAAAAGGCGA AGACTGGATC AACTCCTCTC TTGAAGGTTG ATCTCAAATT AAGAAAACAT 1860 AATGTTATGT TGGCTGGTAA CGTTGTAACT TTCCAGCCGA GTTATCAAAT TGACTTGCCG 1920 ATAGAACATA GTTTCAGCCA CATCTTCTTC GACACTAACC AAATAGGCGC GAGCAATGAT 1980 AGTTCAACCC CGTAATCCAG CCTTATCGGA TGCTATAGAT AGTTTCATAC TATTTACAAG 2040 TAAAATTCGT TTCACATTGA ATCA ATG ATA TTA CAC ACC TAT ATT ATT CTC TCG 2094 Met Ile Leu Hi s Thr Tyr Ile Ile Leu Ser 1 5 10 TTA TTG ACT ATA TTT CCT AAA GCT ATT GGT CTG TCC TTG CAG ATG CCA 2142 Leu Leu Thr Ile Phe Pro Lys Ala Ile Gly Leu Ser Leu Gln Met Pro 15 20 25 ATG GCC TTG GAA GCT AGT TAT GCC TCA TTA GTG GAG AAA GCA ACC CTC 2190 Met Ala Leu Glu Ala Ser Tyr Ala Ser Leu Val Glu Lys Ala Thr Leu 30 35 40 GCT GTT GGA CAA GAA ATT GAT GCC ATA CAA AAG GGT ATT CAG CAA GGT 2238 Ala Val Gly Gln Glu Ile Asp Ala Ile Gln Lys Gly Ile Gln Gln Gly 45 50 55 TGG TTG GAA GTA GAG ACA AGA TTT CCA ACT ATA GTG TCA CAG TTA TCC 2286 Trp Leu Glu Val Glu Thr Arg Phe Pro Thr Ile Val Ser Gln Leu Ser 60 65 70 TAT AGT ACT GGC CCA AAA TTT GCG ATC AAG AAG AAA GAT GCA ACT TTT 2335 Tyr Ser Thr Gly Pro Lys Phe Ala Ile Lys Lys Lys Asp Ala Thr Phe 75 80 85 90 TGG GAT TTC TAT GTT GAA AGT CAA GAG TTG CCA AAC TAC CGG TTA CGC 2382 Trp Asp Phe Tyr Val Glu Ser Gln Glu Leu Pro Asn Tyr Arg Leu Arg 95 100 105 GTG AAA AGG AAC AAT CCC GAA GTT TTG AAA GTT GAT TTT ACT AAA CAG 2430 Val Lys Arg Asn Asn Pro Glu Val Leu Lys Val Asp Phe Thr Lys Gln 110 115 120 TAT AGC GGG TAT TTG GAC GTA GAG GCT GAT GAC AAG CAT TTT TTT TAC 2478 Tyr Ser Gly Tyr Leu Asp Val Glu Ala Asp Asp Lys His Phe Phe Tyr 125 130 135 TGG TTT TTT GAA TCG AGG AAT GAC CCA CAG AAT GAC CCA ATT ATT TTA 2526 Trp Phe Phe Glu Ser Arg Asn Asp Pro Gln Asn Asp Pro Ile Ile Leu 140 145 150 TGG TTG AAT GGT GGC CCT GGC TGC TCT TCC TTG ACT GGT CTC TTT TTC 2574 Trp Leu Asn Gly Gly Pro Gly Cys Ser Ser Leu Thr Gly Leu Phe Phe 155 160 165 170 GAA TTA GGA TCG TCT AGA ATT AAT GAA AAT CTG AAA CCA ATT TTC AAC 2622 Glu Leu Gly Ser Ser Arg Ile Asn Glu Asn Leu Lys Pro Ile Phe Asn 175 180 185 CCC TAT TCG TGG AAT GGT AAT GCT TCA ATC ATC TAC TTA GAT CAA CCG 2670 Pro Tyr Ser Trp Asn Gly Asn Ala Ser Ile Ile Tyr Leu Asp Gln Pro 190 195 200 GTC AAT GTT GGG TTT TCT TAT TCT TCA TCA TCG GTG AGT AAC ACT GTT 2718 Val Asn Val Gly Phe Ser Tyr Ser Ser Ser Ser Val Ser Asn Thr Val 205 210 215 GTT GCG GGA GAA GAT GTG TAT GCA TTT CTT CAG CTT TTT TTT CAA CAC 2766 Val Ala Gly Glu Asp V al Tyr Ala Phe Leu Gln Leu Phe Phe Gln His 220 225 230 TTC CCG GAA TAT CAA ACT AAT GAC TTT CAT ATT GCC GGT GAA TCT TAT 2814 Phe Pro Glu Tyr Gln Thr Asn Asp Phe His Ile Ala Gly Glu Ser Tyr 235 240 245 250 GCA GGA CAT TAC ATT CCG GTG TTT GCA GAC GAA ATT TTG AGT CAA AAG 2862 Ala Gly His Tyr Ile Pro Val Phe Ala Asp Glu Ile Leu Ser Gln Lys 255 260 265 AAC AGA AAT TTC AAT CTT ACT TCA GTC TTG ATC GGA AAT GGA TTA ACT 2910 Asn Arg Asn Phe Asn Leu Thr Ser Val Leu Ile Gly Asn Gly Leu Thr 270 275 280 GAC CCT TTG ACT CAA TAC CGA TAT TAC GAG CCA ATG GCT TGT GGT GAA 2958 Asp Pro Leu Thr Gln Tyr Arg Tyr Tyr Glu Pro Met Ala Cys Gly Glu 285 290 295 GGT GGT GCC CCG TCA GTA CTG CCT GCC GAT GAG TGC GAA AAT ATG CTA 3006 Gly Gly Ala Pro Ser Val Leu Pro Ala Asp Glu Cys Glu Asn Met Leu 300 305 310 GTT ACC CAA GAT AAA TGT TTG TCT TTA ATT CAA GCA TGC TAC GAC TCA 3054 Val Thr Gln Asp Lys Cys Leu Ser Leu Ile Gln Ala Cys Tyr Asp Ser 315 320 325 330 CAG TCG GCA TTC ACA TGC GCA CCG GCT GCC ATT TAT TGT AAT AAC GCT 310 2 Gln Ser Ala Phe Thr Cys Ala Pro Ala Ala Ile Tyr Cys Asn Asn Ala 335 340 345 CAG ATG GGA CCC TAT CAG AGA ACT GGG AAG AAT GTG TAT GAT ATT CGT 3150 Gln Met Gly Pro Tyr Gln Arg Thr Gly Lys Asn Val Tyr Asp Ile Arg 350 355 360 AAG GAA TGT GAT GGT GGA TCC TTG TGC TAT AAG GAC CTT GAA TTC ATC 3198 Lys Glu Cys Asp Gly Gly Ser Leu Cys Tyr Lys Asp Leu Glu Phe Ile 365 370 375 GAT ACC TAC TTA AAT CAA AAG TTT GTT CAA GAT GCT TTG GGC GCC GAG 3246 Asp Thr Tyr Leu Asn Gln Lys Phe Val Gln Asp Ala Leu Gly Ala Glu 380 385 390 GTC GAT ACC TAT GAA TCT TGC AAT TTT GAA ATC AAC AGA AAC TTT TTA 3294 Val Asp Thr Tyr Glu Ser Cys Asn Phe Glu Ile Asn Arg Asn Phe Leu 395 400 405 410 TTT GCT GGA GAT TGG ATG AAA CCT TAT CAT GAA CAT GTC AGC AGT CTC 3342 Phe Ala Gly Asp Trp Met Lys Pro Tyr His Glu His Val Ser Ser Leu 415 420 425 TTG AAC AAA GGT TTG CCC GTT TTG ATT TAC GCA GGG GAC AAA GAT TTC 3390 Leu Asn Lys Gly Leu Pro Val Leu Ile Tyr Ala Gly Asp Lys Asp Phe 430 435 440 ATT TGC AAC TGG TTG GGT AAT CGA GCA TGG ACT GAT GTC TTG CCG TGG 3438 Ile Cys Asn Trp Leu Gly Asn Arg Ala Trp Thr Asp Val Leu Pro Trp 445 450 455 GTT GAT GCT GAT GGT TTT GAA AAA GCC GAA GTC CAA GAT TGG TTG GTT 3486 Val Asp Ala Asp Gly Phe Glu Lys Ala Glu Val Gln Asp Trp Leu Val 460 465 470 AAT GGA AGG AAG GCT GGT GAA TTT AAG AAC TAT AGC AAC TTC ACC TAC 3534 Asn Gly Arg Lys Ala Gly Glu Phe Lys Asn Tyr Ser Asn Phe Thr Tyr 475 480 485 490 CTA AGG GTT TAT GAT GCT GGT CAT ATG GCC CCA TAT GAT CAG CCA GAG 3582 Leu Arg Val Tyr Asp Ala Gly His Met Ala Pro Tyr Asp Gln Pro Glu 495 500 505 AAT TCT CAT GAA ATG GTC AAT AGA TGG ATA TCC GGA GAC TTT AGC TTT 3630 Asn Ser His Glu Met Val Asn Arg Trp Ile Ser Gly Asp Phe Ser Phe 510 515 520 CAC TAG GGTCGCTGAA TTGCAAATAT AGTCAGTAAA TTAAGCCTTG GGGTGCTCTT 3686 His *** 523 TATTGCACGA AAAACTAATA CTTTCAAAGT TGACCATGAG ACTGATTATT GAATTTATAT 3746 TGCTGAATCT GAATTATTGA AATTACACTA TTGTTACTTG TGTAACCATG CTGGTCTGGA 3806 CTGGGCTTGC TGAGTCAGTA CTGTATATCG TACACGGGAA GATC 3850

SEQ ID NO: 6 Sequence Length: 60 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: signal Origin: Organism Name: P. pastoris Strain Name: GTS115 Sequence ATG ATA TTA CAC ACC TAT ATT ATT CTC TCG TTA TTG ACT ATA TTT CCT 48 Met Ile Leu His Thr Tyr Ile Ile Leu Ser Leu Leu Thr Ile Phe Pro 5 10 15 AAA GCT ATT GGT 60 Lys Ala Ile Gly 20

SEQ ID NO: 7 Sequence Length: 57 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: signal Origin: Organism Name: S. cerevisiae Sequence ATG CTT TTG CAA GCT TTC CTT TTC CTT TTG GCT GGT TTT GCA GCC AAA 48 Met Leu Leu Gln Ala Phe Leu Phe Leu Leu Ala Gly Phe Ala Ala Lys 5 10 15 ATA TCT GCA 57 Ile Ser Ala

[Brief description of drawings]

FIG. 1 is a photograph instead of a drawing, which shows an electrophoresis image of a protease isolated from a culture supernatant of a Pichia genus yeast, which was subjected to SDS-PAGE. P: purified serine protease, M: molecular weight marker (SD
S-PAGE Molecular Weight Standards Low; manufactured by Bio-Rad).

FIG. 2 is a photograph instead of a drawing showing an electrophoretic image showing the result of Western blotting using an anti-S. Cerevisiae carboxypeptidase Y antibody against a purified protease derived from the yeast of the genus Pichia. S: S. cerevisiae carboxypeptidase Y P: Purified protease derived from yeast of the genus Pichia M: Molecular weight marker (Pre Stained SDS-PAGE Standards
Low Range; manufactured by Bio-Rad).

FIG. 3 is a diagram showing homology between the N-terminal amino acid sequence of purified Pichia yeast-derived protease and the N-terminal amino acid sequence of baker's yeast-derived carboxypeptidase Y (S-CPY). Numbers are the mature protein N
The number of amino acid residues from the end is shown.

FIG. 4 shows the PRC1 gene encoding S-CPY (S-
It is a figure which shows the plasmid pHO104 in which the PRC1 gene: 2,632 bp was cloned.

FIG. 5 is a diagram showing a restriction enzyme map of a gene (P-PRC1 gene) encoding a protease derived from Pichia yeast.

FIG. 6 is a diagram showing a procedure for constructing a cloning plasmid (pHO110) for the P-PRC1 gene.

FIG. 7 is a diagram showing the homology between the amino acid sequence of Pichia yeast-derived protease (upper row) and the amino acid sequence of S-CPY (lower row). The pre-sequence is italicized, the pro-sequence is shaded and the active center is shown in bold.

FIG. 8: P-PRC1 gene substitution disruption type plasmid pH
It is a figure which shows O115.

FIG. 9 is a diagram showing a procedure for constructing pHO115 (FIG. 1).
(Following 0).

FIG. 10 is a diagram showing a procedure for constructing pHO115, following FIG. 9.

FIG. 11 Eco of DIPY115 strain and GTS115 strain
As a probe by electrophoresing RI digested chromosomal DNA
5 is a photograph as a substitute for a drawing, which shows the result of Southern hybridization using a 0.9 kb EcoRI-PstI fragment of the 5 ′ untranslated region of the P-PRC1 gene. The lane of λ / HindIII shows the results of the marker, the lane of Host shows the GTS115 strain, and the lane of Disruptant shows the result of the disrupted strain DIPY115.

FIG. 12 Eco of DIPY115 strain and GTS115 strain
As a probe by electrophoresing RI digested chromosomal DNA
Fig. 6 is a photograph instead of a drawing, which shows the result of Southern hybridization using a 0.5 kb NsiI-SphI fragment of the coding region of the P-PRC1 gene. The lane of λ / HindIII shows the results of the marker, the lane of Host shows the GTS115 strain, and the lane of Disruptant shows the result of the disrupted strain DIPY115.

FIG. 13 Eco of DIPY115 strain and GTS115 strain
As a probe by electrophoresing RI digested chromosomal DNA
It is a photograph instead of a drawing, which shows the result of Southern hybridization using a 1.2 kb BamHI fragment on the 3'side of the SUC2 gene. The lane of λ / HindIII shows the results of the marker, the lane of Host shows the GTS115 strain, and the lane of Disruptant shows the result of the disrupted strain DIPY115.

FIG. 14: P-PRC1 gene disrupted strain (DIPY115
Strain) and host GTS115 strain genetic maps, their respective degrading activities against Bz-Tyr-pNA (μg / m
It is a figure which shows l).

FIG. 15: DIPY using YPD medium and YPM medium
It is a figure which compared the proliferation degree of each bacterial cell which cultured 115 strain | stump | stock and host GTS115 strain | stump | stock.

FIG. 16 shows a restriction map of plasmid pHO118.

FIG. 17: PP of linearized plasmid pHO118
It is a figure which shows the restriction enzyme map of the overexpression strain integrated in RC1 locus, and the restriction enzyme map of the R-PRC1 locus of the side (recipient) in which linearized plasmid pHO118 is integrated. The probe 1 is AO
The BglII-NsiI fragment of the X1 promoter gene, probe 2 was PstI-Xb of the PRC1 gene.
means an al fragment.

FIG. 18 shows the P-PRC1 gene overexpression strain U8-21 strain (Over-producer) and GTS115 strain (Host) as Pst.
It is a photograph instead of a drawing showing the result of Southern hybridization using probe 1 (BglII-NsiI fragment of AOX1 promoter gene) after digestion with I-BglII. The lane of λ / HindII means a marker.

FIG. 19 shows the P-PRC1 gene overexpressing strain U8-21 strain (Over-producer) and GTS115 strain (Host) as Pst.
It is a photograph instead of a drawing showing the result of Southern hybridization using probe 2 (PstI-Xbal fragment of PRC1 gene) after digestion with I-BglII. The lane of λ / HindII means a marker.

FIG. 20 is a diagram showing a procedure for constructing a secretory expression vector pKM148 of human serum albumin using a pre-peptide sequence of a Pichia yeast-derived protease as a secretory signal.

FIG. 21: Serine protease inhibitor (PMSF) of purified Pichia yeast protease obtained in Example 1
It is a figure which shows the activity inhibition by.

FIG. 22 is a diagram showing activity inhibition of a protease derived from the P-PRC1 gene overexpressing strain U8-21 strain obtained in Example 5 by a serine protease inhibitor (PMSF).

FIG. 23: P-PRC1 gene overexpression strain U8-21
Strain, GTS115 strain and P-PRC1 gene disrupted strain D
It is the figure which compared the protease activity of the culture supernatant (A in the figure) of the IPY115 strain, and the intracellular fraction liquid (B in the figure).

Continuation of front page (51) Int.Cl. ⁶ Identification number Office reference number FI Technology display area // (C12N 9/60 C12R 1:84) (C12N 1/19 C12R 1:84) (C12P 21/02 C12R 1:84) (72) Inventor Noriko Okazaki 2-25-1 Otani Otani, Hirakata City, Osaka Prefecture Midori Cross Central Research Institute Co., Ltd. (72) Satoshi Oya 2-25-1 Otani, Hirakata City, Osaka Prefecture No. Stock Company Midori Cross Central Research Institute (72) Inventor Takao Omura 2-25-1 Otani, Hirakata, Osaka Prefecture Inside Midori Cross Central Research Institute Company

Claims (23)

[Claims] 1. A protease derived from a yeast of the genus Pichia having reactivity with an anti-baker's yeast carboxypeptidase Y antibody. 2. The Pichia yeast-derived protease according to claim 1, which has the following characteristics. (i) Protease activity is inhibited by at least phenylmethylsulfonyl fluoride. (ii) The synthetic substrate benzoyl-L-tyrosine-p-nitroanilide is decomposed to release p-nitroanilide. (iii) having a molecular weight of about 58 kDa (sodium dodecyl sulfate-polyacrylamide gel electrophoresis method) (iv) having four N-type sugar chain binding sites. (V) Protease activity optimum pH is 6-9. (vi) It shows no activity when treated at a pH of about 4 or less or at about 60 ° C. for 1 hour. (vii) Protease activity optimum salt (NaCl) concentration is 0-
1M. 3. The protease derived from the yeast of the genus Pichia according to claim 1 or 2, which has substantially the following amino acid sequence in the N-terminal region. Embedded image 4. The protease derived from yeast of the genus Pichia according to claim 1, which has substantially the following amino acid sequence. Embedded image 5. The precursor before being processed by a protease in a host cell has a prepeptide region, a propeptide region and a protease region, and the prepeptide region has the following amino acid sequence: The protease derived from yeast of the genus Pichia according to any one of claims 1 to 4. Embedded image 6. A precursor before being processed by a protease in a host cell has a prepeptide region, a propeptide region and a protease region, and the propeptide region has the following amino acid sequence: The protease derived from the yeast of the genus Pichia according to any one of claims 1 to 5. [Chemical 4] 7. A DNA containing a nucleotide sequence encoding a protease derived from the yeast of the genus Pichia according to any one of claims 1 to 6. 8. The DNA according to claim 7, wherein the base sequence encoding a protease derived from Pichia yeast has the following base sequence. Embedded image 9. A host cell transformed with the expression vector having the DNA according to claim 7 or 8 is cultured,
Collected from the resulting culture.
7. The method for producing a protease derived from Pichia yeast according to any one of 1 to 6. 10. A DNA containing a nucleotide sequence encoding a protease derived from the yeast of the genus Pichia according to claim 7 or 8.
DNA which is modified so that at least production of a functional product of the DNA is suppressed. 11. The DNA according to claim 10, wherein the modification is insertion of a transformation marker gene into a coding sequence of a DNA containing a nucleotide sequence encoding the protease derived from the yeast of the genus Pichia according to claim 7 or 8. 12. The transformation marker gene, SUC2 gene derived from Saccharomyces cerevisiae, HIS4 gene derived from Pichia pastoris, ARG4 gene, UR
The DNA according to claim 11, which is selected from the group consisting of A3 gene and G418 resistance gene. 13. The DNA according to any of claims 10 to 12.
A modified Pichia yeast strain having a protease-producing ability is suppressed as compared with a natural Pichia yeast strain. 14. A method for producing a heterologous protein, which comprises using the modified Pichia yeast strain according to claim 13 as a host cell. 15. The heterologous protein is human serum albumin, chymase, pro-urokinase-annexin V fusion protein, insulin-like growth factor 1 (IGF).
1) and IGF1 binding protein (IGF
The method for producing a heterologous protein according to claim 14, which is any one selected from the group consisting of 1BP3). 16. A pre-peptide contained in the precursor of the Pichia yeast-derived protease according to any one of claims 1 to 6. 17. The pre-peptide according to claim 16, which is represented by the following amino acid sequence. [Chemical 6] 18. A DNA encoding the pre-peptide according to claim 16 or 17. 19. The method according to claim 18, which is represented by the following nucleotide sequence:
The described DNA. [Chemical 7] 20. A heterologous protein secretory expression vector comprising the DNA according to claim 18 or 19. 21. A heterologous protein secretion expression vector, characterized in that a DNA encoding a heterologous protein is linked downstream of the DNA according to claim 18 or 19. 22. A yeast of the genus Pichia transformed with the expression vector for secreting a heterologous protein according to claim 21. 23. A method for producing a heterologous protein, which comprises culturing the yeast of the genus Pichia according to claim 22 in a medium and collecting the heterologous protein from the resulting medium supernatant.