JPH09261A - Production of glycoprotein - Google Patents

Abstract

PURPOSE: To produce a glycoprotein useful for medicines having a saccharide chain in common with a yeast and a mammalian cell by culturing a modified yeast strain of the genus Pichia having the suppressed saccharide chain extending ability as compared with that of a natural type yeast of the genus Pichia as a host and collecting the resultant product from the cultured product. CONSTITUTION: A modified yeast strain of the genus Pichia having the saccharide chain extending ability suppressed as compared with that of a natural type yeast strain of the genus Pichia by possessing a DNA, having an amino acid sequence of the formula in an N-terminal region and a base sequence capable of coding a protein participating in the extension of the saccharide chain in a glycoprotein derived from the yeast of the genus Pichia and partially modified so as to at least suppress the production of a functional product coded with the DNA is cultured to collect a glycoprotein from the cultured product. Thereby, the objective glycoprotein having a saccharide chain structure which is the same or similar to an endoplasmic reticulum(ER) core saccharide chain in common with the yeast and a mammalian cell and medicinally useful physiological activities is obtained.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention uses a modified Pichia yeast strain having a DNA obtained by modifying a gene encoding a protein involved in sugar chain elongation of a glycoprotein derived from a Pichia yeast as a production strain or a host. The present invention relates to a method for producing a glycoprotein used. This protein is involved in elongation of sugar chains in a glycoprotein produced in a glycoprotein expression system using Pichia yeast as a host, preferably elongation of α-1,6-linked mannose. Most of the physiologically active proteins of pharmaceutical importance are glycoproteins, but according to the method for producing a glycoprotein of the present invention, the Man ₈ GlcNAc ₂ sugar chain having the same structure as the ER core sugar chain common to yeast and mammalian cells is used. Glycoproteins having only can be prepared.

[0002]

BACKGROUND OF THE INVENTION Most proteins that function in vivo are glycoproteins modified with sugar chains. In recent years, regarding the structure of sugar chains, in addition to conventional methods such as analysis using lectins, HPLC, NMR,
New analytical methods and the like using FAB-MAS have been developed, and sugar chain structures of new glycoproteins have been elucidated one after another.
On the other hand, many researchers have also been actively engaged in research on functional analysis of sugar chains, and sugar chains contribute to cell recognition, molecular discrimination, protein structure maintenance and activity, clearance in vivo, secretion,
It has been found that it plays an important role in many in vivo mechanisms such as localization.

For example, in human erythropoietin [see Takeuchi, Proteins / Nucleic Acids / Enzymes, Special Issue “Glycoconjugate” 37, 1713 (1992)], where sugar chain structure and its function are well studied, it is added to erythropoietin. The asparagine-linked sugar chain (Fig. 1 shows its function sharing model),
Neu5Ac (sialic acid) at the tip portion is blood clearance, Gal / GalNAc group (galactose / N-acetylgalactosamine) at the branched portion contributes sterically to binding with the receptor, and the core portion maintains expression of peptide activity. Have been reported to be involved in various functions. As described above, the sugar chain of a glycoprotein has not only a complicated structure but also a wide variety of functions. Therefore, when developing a glycoprotein as a drug, it is important to analyze its structure and function.

By the way, the production of a substance using a microorganism is advantageous in several points as compared with the production of a substance using an animal cell, such as a low production cost and a culture technique cultivated as fermentation technology. is there. However, in microorganisms, sugar chains having the same structure as human glycoprotein (for example, the above sugar chains functioning in erythropoietin described above)
There is a problem that cannot be added. That is,
Glycoproteins derived from animal cells, including humans, are complex type as shown in FIG.
It has a wide variety of mucin-type sugar chains in addition to asparagine-linked sugar chains, but sugar chain addition does not occur in prokaryotic microorganisms such as Escherichia coli, and baker's yeast (Saccharomyces cerevisiae) is a eukaryotic microorganism. However, the only asparagine-linked sugar chain that is added is the high-mannose type, and the mucin-type is added only the sugar chain that contains only mannose as the main component. Therefore, microorganisms are not suitable for the recombinant production of glycoproteins whose sugar chains have important functions such as erythropoietin described above, and in fact, for production of erythropoietin, Chinese hamster ovary cells (CHO
Cells) are used.

[0005] On the other hand, studies have begun to carry out genetic engineering of eukaryotic microorganisms such as baker's yeast by genetic engineering to add sugar chains having the same or similar structure as those of animal cells in baker's yeast cells. There is. In 1994, Schwientek et al. Reported successful expression of human β-1,4-galactosyltransferase gene activity in baker's yeast [Schwientek, T. and Ern.
st, JF, Gene, 145, 299 (1994)]. Also, Krezdrn
Et al. Are also conducting similar research, and in the same manner, human-derived β-1,4-galactosyltransferase and α-2,6-sialyltra were also found in baker's yeast.
nsferase activity expression [Krezdrn, CH, et a
L., Eur. J. Biochem. 220, 809 (1994)].

[0006] On the other hand, attempts have been made to modify the sugar chain itself of the baker's yeast-derived glycoprotein. High-mannose type sugar chains added to baker's yeast-derived glycoproteins contain a larger amount of mannose than the high-mannose type sugar chains of animal cells. Among the added mannose, α-1,6-linked mannose that extends from the α-1,3-linked mannose residue of β-linked mannose and α-1,3-linked mannose added to the outer sugar chain Etc. are structures peculiar to baker's yeast (Fig. 2).

[0007] In 1992, Jigami et al. O of baker's yeast, which is considered to be a key enzyme for elongation of α-1,6-linked mannose.
Successful cloning of CH1 gene (expressing α-1,6-mannosyltransferase) (Nakayama, K., EMBO J.
11, 2511 (1992), see FIG. 2]. The glycoprotein of this OCH1 gene-disrupted strain (Δoch1) contains Man ₈ Glc.
NAc ₂ , Man ₉ GlcNAc ₂ , Man ₁₀ GlcN
Ac ₂ has three kinds of sugar chains added, of which Man is
_{The 8} GlcNAc ₂ sugar chain has the same structure as the ER core sugar chain common to baker's yeast and mammalian cells (the structure described as “Ma” in FIG. 2), Man ₉ GlcNAc ₂ and Man ₁₀
The sugar chain of GlcNAc ₂ has a structure in which α-1,3-linked mannose is added to this ER core sugar chain [Nakanishi-Shindo, Y.,
Nakayama, K., Tanaka, A., Toda, Y. And Jigami, Y., (199
4), J. Biol. Chem.]. Furthermore, △ och1, m
A double mutant of nn1 (see FIG. 2) was prepared and α-1,3 at the end was constructed.
Man with the same structure as the ER core sugar chain common to baker's yeast and mammalian cells by inhibiting binding mannose transfer
A baker's yeast host that added only ₈ GlcNAc ₂ sugar chains could be produced. This Δoch1, mnn1 double mutant strain is considered to be a useful host when a mammalian-derived glycoprotein having a high-mannose type sugar chain is produced by gene recombination technology [Jigami Yoshifumi (1994) Protein・ Nucleic acid / enzyme,
39,657].

By the way, in recent years, Pichia yeast (Pichia pastoris, etc.), which is a methanol-assimilating yeast, has been attracting attention as an effective host for a heterologous protein expression system. The Pichia yeast has a secretory expression amount far higher than that of baker's yeast, and since the culture technique has been established, it is very suitably used as a yeast for industrial production. However, the present situation is that little research has been conducted on the sugar chain elongation mechanism of glycoproteins possessed by Pichia yeast and the sugar chain structure of glycoproteins produced by Pichia yeast.

It is an object of the present invention to provide a method for producing a glycoprotein which uses a yeast of the genus Pichia by a gene recombination technique to produce a glycoprotein having a sugar chain structure which is the same as or similar to a glycoprotein of mammalian origin. And

[0010]

[Means for Solving the Problems] The present inventors have produced various physiologically active proteins using yeast of the genus Pichia as a host. However, as described above, most of the physiologically active proteins are glycoproteins. When Pichia yeast is used as a host for recombinant production, the problem of sugar chains of glycoproteins is an unavoidable problem. Therefore, after conducting various studies to solve such a problem, a gene encoding a protein involved in sugar chain elongation derived from Pichia yeast was successfully cloned, and the protein was expressed in an expression system using the Pichia yeast as a host. ,
The present invention was completed by confirming that it is involved in the elongation of sugar chains of glycoproteins.

That is, according to the present invention, a part of DNA having a nucleotide sequence encoding a protein involved in sugar chain elongation of a glycoprotein is modified so that the production of a functional product encoded by the DNA is at least suppressed. Characterized in that the modified Pichia yeast strain in which the glycoprotein elongation ability of the glycoprotein is suppressed as compared with the natural Pichia yeast strain is cultivated, and the glycoprotein is collected from the culture. To a method for producing a glycoprotein.

The present invention will be described in detail below. (1) Protein involved in sugar chain elongation of glycoprotein This protein is a protein originally produced by yeast of the genus Pichia, which controls the first stage of sugar chain elongation of glycoprotein. It is characterized by having a function of controlling expansion. In addition, in order to simplify the following description, the protein is also referred to as a sugar chain elongation protein.

There are no particular restrictions on the yeast of the genus Pichia from which this sugar chain elongation protein is derived.
a pastoris, Pichia finlandica, Pichia trehalophil
a, Pichia koclamae, Pichia membranaefaciens, Pichi
a opuntiae, Pichia thermotolerans, Pishia salictar
Examples include ia, Pichia guercuum Pichia pijperi and the like. Pichia pastoris (hereinafter referred to as P. pastoris) is preferable.

The sugar chain elongation protein is not particularly limited as long as it is originally derived from yeast of the genus Pichia and has the above-mentioned function, but preferably the amino acid sequence represented by the formula I is added to the N-terminal region. Is a protein that has
More preferably, it is a protein having substantially the amino acid sequence represented by formula II.

[0015]

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[0016]

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The amino acid sequence is partially modified (for example, substitution, deletion, insertion or addition of amino acid residues or peptide chains) within the range in which the above characteristics are not changed.
It may be.

In this sugar chain elongation protein, the amino acid sequence described in formula II, which is exemplified as the primary structure, is a key enzyme for α-1,6-linked mannose elongation derived from baker's yeast, α-1,6-m.
High homology with the amino acid sequence of annosyltransferase (about 4
0%), and the DNA also has a high homology (about 55%) with the OCH1 gene encoding the enzyme derived from baker's yeast as described below. It is likely to be a key enzyme for 1,6-linked mannose elongation.

The sugar chain elongation protein can be produced by culturing 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 cultured cells by a conventional method. it can. In addition, a polypeptide can be synthesized based on the amino acid sequence exemplified in the present invention, or a conventional recombinant DN can be synthesized based on the nucleotide sequence exemplified in the present invention.
It can also be manufactured by the A technology.

(2) DNA Having a Base Sequence Encoding a Sugar Chain Elongating Protein This DNA is characterized by having a base sequence encoding the sugar chain extending protein described above. Hereinafter, this DNA is also referred to as a natural type sugar chain extended DNA in order to distinguish it from the sugar chain extended DNA or the modified sugar chain extended DNA described later. The base sequence is not particularly limited as long as it is a base sequence capable of encoding a sugar chain elongation protein, but is preferably of formula I
The base sequence encoding the amino acid sequence shown by, and more preferably, the base sequence substantially represented by the following formula III is exemplified.

[0021]

[Chemical 6]

The DNA can be produced by a conventionally known method. For example, by using a DNA synthesizer based on the nucleotide sequences exemplified in the present invention, a part or all of D
Synthesizing NA, yeast of the genus Pichia (eg P. pastoris)
It can also be produced by amplification using the chromosomal DNA of No. 1 by the PCR method.

The sugar chain-extended DNA is provided as a gene encoding a protein involved in sugar chain elongation of a glycoprotein, which is produced by a yeast of the genus Pichia, and expresses the glycoprotein using the yeast of the genus Pichia as a host. It is extremely useful for elucidating the mechanism such as the structure and function of sugar chains of glycoproteins in the system.

The sugar chain elongating protein further comprises α- in addition to the core sugar chain of the protein produced by using Pichia yeast as a host.
It has a function of transferring 1,6-linked mannose, and excessively adds mannose as compared with glycoprotein derived from animal cells. Therefore, the reduction or removal of the sugar chain elongation activity originally possessed by the sugar chain elongation protein is such that at least the production of the functional product encoded by the DNA is suppressed in the DNA having the nucleotide sequence encoding the sugar chain elongation protein. Can be achieved by modifying

(3) DNA obtained by modifying natural sugar chain-extended DNA DNA encoding a sugar chain-extended protein of the modified Pichia yeast strain used in the present invention (natural sugar chain-extended DN)
In the modified product of A), a part of the nucleotide sequence of a DNA encoding a protein involved in sugar chain elongation of a glycoprotein derived from a yeast of the genus Pichia has at least suppressed production of a functional product encoded by the DNA. Modified as follows
A.

The term "functional product encoded by DNA" as used herein means a natural sugar chain extension D derived from a yeast of the genus Pichia.
A protein encoded by NA, that is, a protein involved in sugar chain elongation of a glycoprotein, is included in the functional product as long as it has the same function as the above-mentioned protein. Here, "function" refers to a function (activity) relating to sugar chain synthesis / elongation of a sugar chain elongation protein, specifically, "transfer at least α-1,6-linked mannose to core sugar chain" activity (main In the specification, it is referred to as “sugar chain elongation activity”). The term "at least suppress the production of functional products" means not only the case where the protein is not expressed and the protein encoded by the natural type sugar chain-extended DNA of the present invention is not produced, but the product obtained by the expression is a natural type sugar chain. When the function product is not the same as the functional product encoded by the extended DNA and its function is attenuated (that is, when the product has no sugar chain elongation activity possessed by the functional product encoded by the natural type sugar chain extended DNA, and Type sugar chain-extending DNA has a lower activity than the sugar chain-elongating activity possessed by the functional product.

Therefore, the modification mode of DNA is such that the expression of the gene is disabled or the expression / product of the modified sugar chain-extended DNA originally has the sugar chain of the product of the natural type sugar chain-expanded DNA. There is no particular limitation as long as it has no elongation activity or, even if it has, it is attenuated as compared with the sugar chain elongation activity of the product of the natural type sugar chain elongation DNA. Specifically, a modification in which at least one nucleotide in the base sequence of the natural sugar chain-extended DNA is deleted or at least one nucleotide is inserted in the sequence, or a modification of the natural sugar chain-extended DNA Illustrative is a modification in which at least one nucleotide in the base sequence is replaced. Furthermore, addition of at least one nucleotide to the nucleotide sequence of the natural sugar chain-extended DNA is also included in the modification mode. Due to such modification, the reading frame is displaced or the nucleotide sequence is modified, so that 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 natural DNA.

[0028] A preferred modification method is a method of inserting a transformation marker gene into the coding region of the natural sugar chain-extended DNA. According to this, it is possible to destroy the natural type sugar chain-extended DNA and to easily screen the mutant having the modified type sugar chain-extended DNA by using the introduced transformation marker gene as an index. There are advantages. In addition to the transformation marker gene, the gene for the glycoprotein to be produced can be inserted. According to this, the sugar chain extension D
Modification of NA and expression of the glycoprotein to be produced can be performed simultaneously by a single operation.

Examples of the transformation marker gene used include the HIS4 gene of P. pastoris or baker's yeast, AR
G4 gene, URA3 gene, SUC2 gene, G41
8 resistance gene etc. are illustrated. The HIS4 gene is preferred. The gene for the glycoprotein is not particularly limited as long as it is the DNA of the desired glycoprotein to be produced. Specifically, it is a soluble high-affinity IgE receptor α chain (sFcεRIα, JP-A-6-169776). ),
Interferon α (JP-A 61-185189), urokinase (JP-A 60-180591), chymase [Caughey, GH, et al., J. Biol. Chem.
266, 12956 (1991)], urinary trypsin inhibitor [Kaumeyer, JF, et al., Nucleic Acids Res. 14,78.
39 (1986)], and IGF binding protein (IGF1BP3, Japanese Patent Laid-Open No. 3-505397).

(4) Modified yeast strain belonging to the genus Pichia The modified yeast strain belonging to the genus Pichia used in the present invention has a sugar chain elongation as compared with a yeast strain belonging to the genus Pichia on the basis of having the above-described modified sugar chain-extended DNA. It is a yeast strain of the genus Pichia with suppressed ability. That is, it is a yeast of the genus Pichia having the above modified sugar chain-extended DNA instead of the natural sugar chain-extended DNA, and the activity of the functional product encoded by the natural sugar chain-extended DNA is attenuated or the activity is Not expressed.

Such a modified Pichia yeast strain can be prepared by various methods. For example, modification of a natural sugar chain-extending DNA in a natural Pichia yeast or random mutation in a natural Pichia yeast strain, resulting in a sugar chain elongation activity higher than that of a natural Pichia yeast strain. Examples include a method of selecting a mutant that is suppressed. A method of modifying a natural sugar chain-extended DNA in a natural yeast of the genus Pichia is preferably used. A method for producing a modified Pichia yeast strain by modifying a natural sugar chain-extended DNA is specifically, a method of introducing a DNA that transduces at a specific locus of the natural sugar chain-extended DNA by a site-specific integration method. Be implemented. The transduced DNA is integrated by replacing the endogenous native DNA of the host. A convenient method for introducing the transducing DNA into the target locus of the yeast host is to delete the inside of the target gene DNA fragment, or select marker gene DNA or heterologous gene expression DNA.
To produce a linear DNA fragment into which the fragment has been inserted. This causes transformation to direct the expression product to homologous recombination at specific sites in the DNA that affect sugar chain elongation activity.

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. Cell. Biol., 5,3376 (1985), U.S. Pat.No. 4,879,23.
No. 1], 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. Examples of suitable methanol-assimilating yeasts include auxotrophic P. pastoris GTS115 strain (NRRL Y-15851), P. pastoris GS19.
0 strain (NRRL Y-18014), P. pastoris PP
F1 strain (NRRL Y-18017), wild type P. pastor
is strain (NRRL Y-11430, NRRL Y-11
431) etc. are illustrated.

Further, more preferably, a strain lacking at least one autotrophic marker gene, for example, HIS4 deficient P. pastoris GS115 strain (ATCC2
0864), ARG4 deficient P. pastoris GS190 strain,
HIS4 / URA3 deficient P. pastoris GS4-2 strain, H
IS4 / URA4 deficient P. pastoris PPF1 strain (NRR
LY-18017: U.S. Pat. No. 4,812,405) and the like. Thus, when the host cell is a strain lacking at least one autotrophic marker gene,
As the DNA to be transduced, it is preferable to use one having an autotrophic marker gene that is deleted in the host cell. According to such a method, a transformant (modified Pichia yeast strain) in which transducing DNA has been incorporated and sugar chain-extended DNA has been modified can be identified and selected quickly and easily.

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 sugar chain-extended DNA does not affect the growth of Pichia yeast under nutritional conditions. Therefore, the modified Pichia yeast strain used in the present invention serves as an excellent production strain that produces a pharmaceutically useful glycoprotein or a host for producing such a production strain. That is, the yeast has a decreased or disappeared sugar chain elongation ability derived from the host cell as compared with the natural Pichia yeast strain, and therefore, a glycoprotein produced by a mammalian cell, particularly a human-derived cell. Glycoproteins having the same or similar sugar chain structure can be produced.

The glycoprotein having the same or similar sugar chain structure as the glycoprotein produced by the above-mentioned mammalian cells, especially cells derived from humans, is a glycoprotein having a sugar chain structure on the protein molecule. Although not particularly limited, preferably a pharmaceutically useful physiologically active protein, specifically, a soluble high affinity IgE receptor α chain (sFcεRIα), epidermal growth factor (EGF), growth hormone releasing factor (GR
F), IGF1 binding protein 3 (IGF1BP3), pro-urokinase-annexin V fusion protein, chymase, urinary trypsin inhibitor and the like.

Expression systems useful for glycoprotein production can be prepared by various methods. For example, a method of introducing a DNA encoding a glycoprotein into the modified Pichia yeast strain described above, a DN encoding a glycoprotein together with a transformation marker gene in the base sequence of a natural sugar chain-extended DNA.
A method for transforming a native Pichia yeast using the DNA having A inserted therein, a natural sugar chain-extended DNA possessed by a recombinant Pichia yeast strain having a DNA encoding a glycoprotein, and the modified sugar chain Examples thereof include a method of mutating to an extended DNA mode, a method of simultaneously transforming a natural yeast strain of the genus Pichia with the above modified sugar chain extended DNA and DNA encoding a glycoprotein, and the like.

The yeast of the genus Pichia of the recombinant glycoprotein expression system is
It has at least a promoter region, a DNA encoding substantially the desired glycoprotein, and a transcription terminator region in the direction of the reading frame of transcription. These DNAs are RNs that encode the desired glycoprotein.
As transcribed into A, 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
Examples thereof include a promoter for the oris aldehyde dehydrogenase gene or a promoter for the folate dehydrogenase gene of P. pastoris. Preferably, P. pastoris AOX1 promoter (Ellis et a
L., Mol. Cell. Biol., 5,111 (1985), U.S. Patent No. 4,855,23.
No. 1), and more preferably, a mutant AOX2 promoter (Ohi,
H et al., Mol. Gen. Genet., 243, 489-499, 1994, JP-A-4-299984).

The DNA encoding the secretory signal sequence may be provided before the DNA encoding substantially the desired glycoprotein. According to the recombinant glycoprotein expression system having such DNA, the glycoprotein is secreted and produced outside the host cell, so that the desired glycoprotein can be easily isolated and purified. As the DNA encoding the secretory signal sequence, a DNA encoding a natural secretory signal sequence related to glycoprotein, a DNA encoding a baker's yeast α-mating factor (αMF) signal sequence (coding a processing site, Lys-Arg), which contains the DNA sequence
DNA encoding a signal sequence such as bovine lysozyme C signal sequence that functions in a methanol-assimilating yeast cell
And the like.

The transcription terminator may be one having a subsegment that provides a transcription termination signal for transcription from the promoter, may be the same as or different from the gene of the promoter source, and may be a glycoprotein. It may be obtained from the encoding gene.

The expression system used in the present invention is the above-mentioned DN.
In addition to the A sequence, it may further contain a selectable marker gene. The selectable marker gene used is HI
S4, ARG4, URA3, baker's yeast SUC2, G41
8 resistance genes and the like.

The modified Pichia yeast strain transformed to the desired phenotype can produce glycoprotein by culturing by a method commonly used in the art. The medium used is not particularly limited, and a normal natural medium (YPD
Medium, YPM medium) and the like. The culture temperature is preferably a temperature suitable for the growth of Pichia yeast host cells and the production of the desired glycoprotein, and is about 20 to 30 ° C, preferably about 23 to 25 ° C. As the pH of the medium, a pH suitable for host cell growth and production of a desired glycoprotein can be appropriately adopted. Further, aeration and stirring can be added if necessary. After culturing, collect the culture supernatant,
A desired heterologous protein can be purified and obtained from the supernatant by a method known per se, for example, fractionation method, ion exchange, gel filtration, hydrophobic interaction chromatography or affinity column chromatography.

[0044]

INDUSTRIAL APPLICABILITY According to the present invention, a modified Pichia yeast strain having a growth ability equivalent to that of a natural Pichia yeast strain and having a decreased or eliminated sugar chain elongation ability as compared with the natural Pichia yeast strain. By using as a production strain or a host, a pharmaceutically useful glycoprotein having a sugar chain structure that is the same as or similar to the ER core sugar chain common to yeast and mammalian cells can be prepared. According to the present invention, since the glycoprotein is produced by using the yeast of the genus Pichia, which has a higher secretory expression amount than the baker's yeast, the production amount can be improved in the production of the glycoprotein using the yeast.

[0045]

The present invention will be described in more detail based on the following examples and reference examples. However, the present invention is not limited to this. 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 familiar with the procedures used for cloning DNA, 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.

Example (a) Acquisition of gene for sugar chain elongation protein derived from yeast of the genus Pichia The sugar chain elongation gene OCH1 derived from baker's yeast (Saccharomyces cerevisiae) was amplified by the PCR method to form a probe, which was used as a chromosomal gene of the yeast of the genus Pichia. By a Southern analysis to encode a protein involved in sugar chain elongation derived from yeast of the genus Pichia
I searched for A.

(1) OCH1 of baker's yeast by PCR method
Amplification and acquisition of gene In order to clone the sugar chain elongation gene OCH1 derived from baker's yeast, the literature [The EMBO Journal vol.11 no.7 p2511-2
519 (1992): p2513, Fig. 2], based on the DNA sequence disclosed herein, an N-terminal primer having a HindIII recognition site added to a sequence complementary to both terminal DNAs of the protein translation region:
5'-CGAAGCTTATGTCTAGGAAGTT
GTCCCCACCTG-3 ′ and C-terminal side primer:
5'-CGAAGCTTTATTTATGACCTGCA
TTTTTTACAG-3 '(primer for PCR amplification) is a DNA synthesizer (ABI, model 392DN).
A / RNA synthesizer) was used for chemical synthesis. Using this primer, the conventional method (Sherman, F., Fink, GR
and Hicks, JB (1986) Laboratory course manual f
or methods in yeast genetics, Cold Spring Harbor L
aboratory, Cold Spring Harbor, New York) baker's yeast AH22 strain (a, len2, his4, can1) (Hinn
en, A. et al (1978) Proc. Natl. SciUSA 75, p. 1929
) -Derived chromosomal DNA as a template for PCR reaction (94
1 minute at 50 ° C., 2 minutes at 50 ° C., 2 minutes at 72 ° C./25 cycles) [DNA Thermal Cycler Model PJ2000, Perkin
-Elmer]. As a result of agarose gel electrophoresis of the amplified DNA fragment, a clear single band was observed on the gel. The amplified DNA fragment showed the size expected from the set primer (1458 bp).

(2) Subcloning of OCH1 gene derived from baker's yeast The PCR amplified fragment obtained in (1) was digested with HindIII and then subcloned into the HindIII site of pUC19. The prepared plasmid (pKM049, FIG. 3) was prepared from several kinds of restriction enzymes (BamHI, EcoRI, Kpn).
OC that has been digested with I) and its cleavage pattern has been announced
H1 gene cleavage site [EMBO J. 11, 7 p2511-2519 (19
92): p2512, Fig.1 and p2513, Fig.2], and they were in perfect agreement.

(3) Southern hybridization of chromosomal gene of Pichia yeast using OCH1 gene as a probe Y of Pichia pastoris (Pichia pastoris GTS115 strain)
Incubate in PD medium (1% yeast extract, 2% peptone, 2% glucose) at 30 ℃ for 3 days.
man et al.'s method (Sherman, F., Fink, GR and Hicks, J.
B. (1986) Laboratory course manual for methods in
yeast genetics, Cold Spring Harbor Laboratory, Col
Chromosomal DNA was prepared according to D Spring Harbor, New York). The obtained chromosomal DNA was treated with various restriction enzymes and then subjected to agarose gel electrophoresis, and the DNA fragment was transferred to a nylon membrane (Hybond-N, manufactured by Amersham). Baker's yeast-derived O obtained in (1)
The CH1 gene HindIII fragment was labeled with "DIG-ELISA.
Labeling kit "(manufactured by Boehringer Mannheim) is used as a probe, and Southern hybridization is performed by a conventional method (Sambrook, J., Fritsh, ef and M
aniatis, T. (1989) Molecular cloning: A laboratory
manual, Cold Spring Harbor Laboratory, Cold Sprin
g Harbor, New York), and whether or not there is a gene having homology with the baker's yeast-derived OCH1 gene.

Since the homology between the baker's yeast-derived OCH1 gene and the chromosomal DNA of the yeast of the genus Pichia is unknown, the hybridization temperature (65 ° C, 55 ° C,
45 ° C.) and washing conditions (salt concentration: 0.2-0.5 × SS)
C, temperature: room temperature to 42 ° C.). As a result, hybridization was performed overnight at 55 ° C and 2
× SSC, room temperature, 30 minutes, washed twice, then 0.5 ×
EcoRI when washed twice in SSC, 42 ℃, 30 minutes
A clear band of about 5 kb was observed for the digest. It was suggested that a gene having a homology with the OCH1 gene derived from baker's yeast is present on the chromosome of the yeast of the genus Pichia by making the hybridization temperature and washing conditions mild as described above.

(4) Construction of λgt10 library Based on the result of (3), chromosomal DNA of yeast of the genus Pichia
The EcoRI fragment (about 5 kb) was cloned. First, about 150 μg of chromosomal DNA derived from P. pastoris GTS115 strain (NRRL deposit number Y-15851)
Was digested with 200 enzyme units of EcoRI overnight and then 0.
About 4.5-6 kb by 8% agarose gel electrophoresis
Was separated and collected. Part of recovered DNA is 1μ
λgt10arm of g (“lambda gt10 vector digeste
d with EcoRI and dephosphorylated ", manufactured by Stratagene), GigapackII Gold Packag
Packaging was performed using ing Extract (manufactured by Stratagene). As a result, the number of plaques required for screening was obtained.

(5) Plaque Hybridization The recombinant λ phage library prepared in (4) was titrated to 200 to 300 plaques per plate of 80 mm diameter, and a nylon membrane filter (Hybond-N, Amersham) was used. (Made by the company).
Ten of these filters were prepared (total screening number: about 3000 plaques), and hybridization was carried out using the baker's yeast-derived OCH1 gene fragment as a probe. As a result, 14 clear positive plaques were detected.

(6) Purification of λDNA Among the positive plaques detected in (5), 10 plaques were arbitrarily selected, and after single plaque isolation,
ΛDNA was extracted and purified using Sephaglas ^™ PhagePrep Kit (Pharmacia). Each purified DNA was digested with several restriction enzymes (EcoRI, BglII, HindIII,
When the digestion patterns of (XhoI) were compared by agarose gel electrophoresis, it was found that up to 8 out of 10 clones had the same insert DNA (about 5 kb).

(7) Subcloning About one of the clones, the inserted EcoRI
The fragment was subcloned into the EcoRI site of pUC19 to create pKM50 (Figure 4).

(8) Determination of nucleotide sequence and amino acid sequence of DNA fragment inserted into pKM50 Eco derived from yeast belonging to the genus Pichia inserted into pKM50
The base sequence of the RI fragment was determined. A restriction enzyme map of the chromosomal DNA fragment cloned using pKM50 was prepared and further detailed analysis was performed using several restriction enzymes. As a result, a homologous region with the baker's yeast OCH1 gene was found in a BglII fragment of about 2.5 kb. Has been shown to exist. Therefore, the DNA of this BglII fragment of about 2.5 kb
The base sequence was determined. Specifically, the insert fragment was subcloned into pUC19 using several restriction enzymes as subfragments, and their DNA base sequences were analyzed using M13-40 primer and Reverse primer (Pharmacia LKB Bi-Technology). (A.
L. F. DNA sequencer, Pharmacia LKB Bi-Technology).

Baker's yeast OCH1 inserted in pKM50
A gene fragment containing a region showing homology with a gene [BglII
~ SalI fragment (about 3.0 kb)] was determined to have an open reading frame consisting of 404 amino acids.
e (ORF) (Fig. 4, shaded area) was present. BglII
~ Nucleotide sequence up to SalI site (2858bp) and Op
The sequence obtained by translating the en Reading Frame region into amino acids is shown in SEQ ID NO: 1 in the sequence listing. In this region, a site (Asn-Xa) where asparagine-linked sugar chain addition may occur.
There were two a-Ser / Thr).

Next, the amino acid sequence of the ORF region derived from the yeast of the genus Pichia was compared with the amino acid sequence of the protein derived from the OCH1 gene derived from the baker's yeast. As a result, the EcoRI fragment (about 5 k
The amino acid sequence encoded by b) had about 40% homology with the amino acid sequence encoded by the baker's yeast-derived OCH1 gene (FIG. 5). The homology at the DNA level encoding the amino acid sequence is about 55.
%Met. Regarding the asparagine-binding sugar chain addition site surrounded by □ in FIG. 5, agreement was observed in only one homologous region (Asn ¹⁹⁹ and bread of the sugar chain elongation protein of the present invention derived from the yeast of the genus Pichia). As of yeast OCH1 protein
n ²⁰³ ). The molecular weight predicted from the amino acid sequence was 55 kDa for baker's yeast OCH1 protein, but 46 kDa for sugar chain elongation protein derived from yeast of the genus Pichia.

Next, the hydrophobicity of both proteins was compared. As a result, both showed very similar patterns as shown in FIG. From this, the EcoRI fragment obtained from the Pichia yeast is OC derived from the Pichia yeast.
It was suggested to be the H1 gene. In addition, baker's yeast O
The CH1 protein has a transmembrane region (membrane spa) near the N-terminus.
Although hydrophobic region seems nning domain) is present (Thr ¹⁶ ~Phe ^30), further long hydrophobic region than a sugar chain elongation protein from yeast of the genus Pichia was present.

(B) Preparation of sugar chain-extended DNA-disrupted strain (1) Genomic S of sugar chain-extended DNA derived from yeast of the genus Pichia
Outhern Hybridization Analysis For the purpose of producing a strain in which the sugar chain-extended DNA derived from the yeast of the genus Pichia cloned in (a) above was disrupted, the D
Genomic Southern Hybridization analysis was performed to confirm that NA is a single gene on the chromosome. The chromosome of the P. pastoris GTS115 strain used as a host was digested with restriction enzymes BglII, EcoRI, SphI, and XbaI, subjected to agarose gel electrophoresis, and then blotted on a nylon membrane. Next, D containing a DNA sequence encoding the protein translation region of the sugar chain extension DNA derived from the yeast of the genus Pichia
NA fragment (Fig. 4, HnidIII-HincII of pK50)
Hybridization was carried out using a fragment of about 900 bp, a region of base number 1488 to base number 2385 in the base sequence table SEQ ID NO: 1) as a probe. FIG. 7 shows the results. As can be seen, sugar chain extension DNA derived from Pichia yeast
The probe hybridized only to a single band with either restriction enzyme. From the above results, it was found that the sugar chain extended DNA derived from the yeast of the genus Pichia is a single gene.

(2) Preparation of Pichia yeast-derived sugar chain-extended DNA-disrupted strain using HIS4 as a selectable marker Plasmid pKM50 (see FIG. 4) containing the Pichia yeast-derived sugar chain extended DNA and the surrounding chromosomal fragment Asu
The II and BalI sites were digested to blunt ends and the HIS4 gene and soluble high affinity IgE receptor α chain gene (sFcεRIα) expression unit were inserted between them to create plasmid pKM74 (FIG. 8). The soluble high-affinity IgE receptor α chain gene (sFcεRIα) expression unit is a signal sequence of the baker's yeast SUC2 gene, and the sFcεRIα gene [Nucleic Acids Research, Vo
lume 16 Number 8, 3584 (1988)] and a promoter region of P. pastoris AOX2 gene and a P. pastoris AOX1 gene terminator region are ligated to the mature N-terminal. It is capable of secretory expression of the extracellular region (172 amino acids) of the affinity IgE receptor.

The pKM74 was digested with SphI and PstI to obtain P. pastoris GTS115 strain (his4) (NRRL
When deposited number Y-15851) was transformed, 45
A transformant (HIS4) of the strain could be obtained. Therefore, some of these transformants were selected and the following analysis was performed.

(3) Analysis of GTS115 / pKM74 Transformant Regarding baker's yeast OCH1 gene-disrupted strain, it has been reported that the strain has lost high temperature resistance and cannot grow at 37 ° C. [Nakayama, K., et. al. EMBO J. 11, 2511 (199
2)]. Therefore, the temperature sensitivity of the transformant obtained in (2) was examined. When 45 strains were observed using the YPD plate for growth at 25 ° C., 30 ° C. and 37 ° C., respectively, 10 strains could not grow at 37 ° C. On the other hand, arbitrarily select 10 transformants and
When nomic Southern Hybridization analysis was carried out, sugar chain-extended DNA of two of these strains (KM74-2 and KM74-5 strain) was disrupted. These two strains are both 3
I couldn't grow at 7 ℃, so I couldn't grow temperature and Genomic Southern H
The ybridization analysis was shown to be consistent.

Further, a more detailed Genomic Southern Hybridization analysis was performed on the transformed strain (KM74-2 strain) (FIG. 9). The KM45 strain shown in FIG. 9 is pKM7.
A transformant in which the HIS4 gene of No. 4 and a soluble high-affinity IgE receptor α chain gene (sFcεRIα) expression unit DNA fragment are integrated into the his4 gene locus of P. pastoris GTS115his4 strain and capable of secreting and expressing the sFcεRI α chain protein Is. GTS115 strain, OCH1 gene wild strain KM45 strain, OCH1 gene disruption strain KM74
-2 strain, chromosomal DNA was EcoRI and Bgl
After digestion with II, the upstream region of the sugar chain-extended DNA (probe 1: pKM50 BglII-AsuII fragment 1256 bp shown in FIG. 4, base number 2 to base number 1258 shown in SEQ ID NO: 1 in the nucleotide sequence table in FIG. 4) and sugar chain Region of extended DNA (Fig. 9)
Inside, probe 2: pKM50, HindIII shown in FIG.
-EcoT14I fragment 468 bp, Genomic Southern Hybridization analysis was carried out using as a probe the base number 1488 to the base number 1948 described in SEQ ID NO: 1 of the base sequence table, and KM.
It was confirmed that the sugar chain-extended DNA of strain 74-2 was destroyed by the introduced pKM74 gene fragment (Fig. 1).
0, see FIG. 11).

(C) Analysis of sFcεRIα chain protein produced by a sugar chain-expanded DNA-disrupted strain of yeast Pichia To investigate sugar chain addition of a sugar chain-expanded DNA-disrupted strain of Pichia yeast, a sugar chain-expanded DNA-disrupted strain KM74-2 And KM7
4 × 5 strain and KM45 strain as a wild type strain 3 × YP + 2%
Methanol medium (3% yeast extract, 6%
After culturing in bactopeptone, 2% methanol) at 25 ° C. for 4 days, sFcεRIα chain protein was purified from the culture supernatant by an IgE affinity column. Each purified sF
Samples obtained by removing asparagine-linked sugar chains with cεRI α-chain protein and PNGaseF (manufactured by Genzyme)
It was analyzed by DS-polyacrylamide gel electrophoresis (Fig. 12). As a result, a high molecular weight sFcεRIα chain protein was observed in the KM45 strain in which the sugar chain-extended DNA was not destroyed (FIG. 12, lane 1), whereas the sugar chain-extended DNA was
S derived from the disrupted strains KM74-2 and KM74-5
In the FcεRI α-chain protein, the elongation of the sugar chain was suppressed, so that the protein molecular species showing high molecular weight disappeared (Fig. 1).
2, lanes 2, 3). Furthermore, the sugars of these proteins are PN
When removed with GaseF (manufactured by Genzyme), they showed the same molecular weight (FIG. 12, lanes 4, 5, 6), and it was confirmed that the difference in the molecular weight distribution was due to the sugar chain. From the above results, it was suggested that the sugar chain elongation was suppressed in the P. pastoris sugar chain elongation DNA-disrupted strain.

[0065]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 2858 Sequence type: Nucleic acid Number of strands: 2 Topology ─: Linear Sequence type: genomic DNA Origin organism name: P. pastoris Strain name: GTS115 Sequence characteristics: Characterize symbol: CDS existing position: 1027-2238 method to determine the characteristics: S, P sequences AGATCTGCCT GACAGCCTTA AAGAGCCCGC TAAAAGACCC GGAAAACCGA GAGAACTCTG 60 GATTAGCAGT CTGAAAAAGA ATCTTCACTC TGTCTAGTGG AGCAATTAAT GTCTTAGCGG 120 CACTTCCTGC TACTCCGCCA GCTACTCCTG AATAGATCAC ATACTGCAAA GACTGCTTGT 180 CGATGACCTT GGGGTTATTT AGCTTCAAGG GCAATTTTTG GGACATTTTG GACACAGGAG 240 ACTCAGAAAC AGACACAGAG CGTTCTGAGT CCTGGTGCTC CTGACGTAGG CCTAGAACAG 300 GAATTATTGG CTTTATTTGT TTGTCCATTT CATAGGCTTG GGGTAATAGA TAGATGACAG 360 AGAAATAGAG AAGACCTAAT ATTTTTTGTT CATGGCAAAT CGCGGGTTCG CGGTCGGGTC 420 ACACACGGAG AAGTAATGAG AAGAGCTGGT AATCTGGGGT AAAAGGGTTC AAAAGAAGGT 480 CGCCTGGTAG GGATGCAATA CAAGGTTGTC TTGGAGTTTA CATTGACCAG ATGATTTGGC 540 TTTTTCTCTG TTCAATTCAC ATTTTTCAGC GAGAATCGGA TTGACGGAGA AATGGCGGG G 600 TGTGGGGTGG ATAGATGGCA GAAATGCTCG CAATCACCGC GAAAGAAAGA CTTTATGGAA 660 TAGAACTACT GGGTGGTGTA AGGATTACAT AGCTAGTCCA ATGGAGTCCG TTGGAAAGGT 720 AAGAAGAAGC TAAAACCGGC TAAGTAACTA GGGAAGAATG ATCAGACTTT GATTTGATGA 780 GGTCTGAAAA TACTCTGCTG CTTTTTCAGT TGCTTTTTCC CTGCAACCTA TCATTTTCCT 840 TTTCATAAGC CTGCCTTTTC TGTTTTCACT TATATGAGTT CCGCCGAGAC TTCCCCAAAT 900 TCTCTCCTGG AACATTCTCT ATCGCTCTCC TTCCAAGTTG CGCCCCCTGG CACTGCCTAG 960 TAATATTACC ACGCGACTTA TATTCAGTTC CACAATTTCC AGTGTTCGTA GCAAATATCA 1020 TCAGCC ATG GCG AAG GCA GAT GGC AGT TTG CTC TAC TAT AAT CCT CAC AAT 1071 Met Ala Lys Ala Asp Gly Ser Leu Leu Tyr Tyr Asn Pro His Asn 1 5 10 15 CCA CCC AGA AGG TAT TAC TTC TAC ATG GCT ATA TTC GCC GTT TCT GTC 1119 Pro Pro Arg Arg Tyr Tyr Phe Tyr Met Ala Ile Phe Ala Val Ser Val 20 25 30 ATT TGC GTT TTG TAC GGA CCC TCA CAA CAA TTA TCA TCT CCA AAA ATA 1167 Ile Cys Val Leu Tyr Gly Pro Ser Gln Gln Leu Ser Ser Pro Lys Ile 35 40 45 GAC TAT GAT CCA TTG ACG CTC CGA TCA CTT GAT TTG AAG ACT TTG GAA 1215 Asp Tyr Asp Pro Leu Thr Leu Arg Ser Leu Asp Leu Lys Thr Leu Glu 50 55 60 GCT CCT TCA CAG TTG AGT CCA GGC ACC GTA GAA GAT AAT CTT CGA AGA 1263 Ala Pro Ser Gln Leu Ser Pro Gly Thr Val Glu Asp Asn Leu Arg Arg Arg Arg 65 70 75 CAA TTG GAG TTT CAT TTT CCT TAC CGC AGT TAC GAA CCT TTT CCC CAA 1311 Gln Leu Glu Phe His Phe Pro Tyr Arg Ser Tyr Glu Pro Phe Pro Gln 80 85 90 95 CAT ATT TGG CAA ACG TGG AAA GTT TCT CCC TCT GAT AGT TCC TTT CCG 1359 His Ile Trp Gln Thr Trp Lys Val Ser Pro Ser Asp Ser Ser Phe Pro 100 105 110 AAA AAC TTC AAA GAC TTA GGT GAA AGT TGG CTG CAA AGG TCC CCA AAT 1407 Lys Asn Phe Lys Asp Leu Gly Glu Ser Trp Leu Gln Arg Ser Pro Asn 115 120 125 TAT GAT CAT TTT GTG ATA CCC GAT GAT GCA GCA TGG GAA CTT ATT CAC 1455 Tyr Asp His Phe Val Ile Pro Asp Asp Ala Ala Trp Glu Leu Ile His 130 135 140 CAT GAA TAC GAA CGT GTA CCA GAA GTC TTG GAA GCT TTC CAC CTG CTA 1503 His Glu Tyr Glu Arg Val Pro Glu Val Leu Glu Ala Phe His Leu Leu 145 150 155 CCA GAG CCC ATT CTA AAG GCC GAT TTT TTC AGG TAT TTG ATT CTT TTT 155 1 Pro Glu Pro Ile Leu Lys Ala Asp Phe Phe Arg Tyr Leu Ile Leu Phe 160 165 170 175 GCC CGT GGA GGA CTG TAT GCT GAC ATG GAC ACT ATG TTA TTA AAA CCA 1599 Ala Arg Gly Gly Leu Tyr Ala Asp Met Asp Thr Met Leu Leu Lys Pro 180 185 190 ATA GAA TCG TGG CTG ACT TTC AAT GAA ACT ATT GGT GGA GTA AAA AAC 1647 Ile Glu Ser Trp Leu Thr Phe Asn Glu Thr Ile Gly Gly Val Lys Asn 195 200 205 AAT GCT GGG TTG GTC ATT GGT ATT GAG GCT GAT CCT GAT AGA CCT GAT 1695 Asn Ala Gly Leu Val Ile Gly Ile Glu Ala Asp Pro Asp Arg Pro Asp 210 215 220 TGG CAC GAC TGG TAT GCT AGA AGG ATA CAA TTT TGC CAA TGG GCA ATT 1743 Trp His Asp Trp Tyr Ala Arg Arg Ile Gln Phe Cys Gln Trp Ala Ile 225 230 235 CAG TCC AAA CGA GGA CAC CCA GCA CTG CGT GAA CTG ATT GTA AGA GTT 1791 Gln Ser Lys Arg Gly His Pro Ala Leu Arg Glu Leu Ile Val Arg Val 240 245 250 255 GTC AGC ACG ACT TTA CGG AAA GAG AAA AGC GGT TAC TTG AAC ATG GTG 1839 Val Ser Thr Thr Leu Arg Lys Glu Lys Ser Gly Tyr Leu Asn Met Val 260 265 270 GAA GGA AAG GAT CGT GGA AGT GAT GTG ATG GAC TGG ACG GGT CCA GGA 1887 Glu Gly Lys Asp Arg Gly Ser Asp Val Met Asp Trp Thr Gly Pro Gly 275 280 285 ATA TTT ACA GAC ACT CTA TTT GAT TAT ATG ACT AAT GTC AAT ACA ACA 1935 Ile Phe Thr Asp Thr Leu Phe Asp Tyr Met Thr Asn Val Asn Thr Thr 290 295 300 GGC CAC TCA GGC CAA GGA ATT GGA GCT GGC TCA GCG TAT TAC AAT GCC 1983 Gly His Ser Gly Gln Gly Ile Gly Ala Gly Ser Ala Tyr Tyr Asn Ala 305 310 315 TTA TCG TTG GAA GAA CGT GAT GCC CTC TCT GCC CGC CCG AAC GGA GAG 2031 Leu Ser Leu Glu Glu Arg Asp Ala Leu Ser Ala Arg Pro Asn Gly Glu 320 325 330 335 ATG TTA AAA GAG AAA GTC CCA GGT AAA TAT GCA CAG CAG GTT GTT TTA 2079 Met Leu Lys Glu Lys Val Pro Gly Lys Tyr Ala Gln Gln Val Val Leu 340 345 350 TGG GAA CAA TTT ACC AAC CTG CGC TCC CCC AAA TTA ATC GAC GAT ATT 2127 Trp Glu Gln Phe Thr Asn Leu Arg Ser Pro Lys Leu Ile Asp Asp Ile 355 360 365 CTT ATT CTT CCG ATC ACC AGC TTC AGT CCA GGG ATT GGC CAC AGT GGA 2175 Leu Ile Leu Pro Ile Thr Ser Phe Ser Pro Gly Ile Gly His Ser Gly 370 375 380 GCT GGA GAT TTG AAC CAT CAC CTT GCA TAT ATT AGG CAT ACA TTT GAA 2223 Ala Gly Asp Leu Asn His His Leu Ala Tyr Ile Arg His Thr Phe Glu 385 390 395 GGA AGT TGG AAG GAC TAA AGAAAGCTAG AGTAAAATAG ATATAGCGAG 2271 Gly Ser Trp LysGAp *** 400 AT GAATACCTTC TTCTAAGCGA TCGTCCGTCA TCATAGAATA TCATGGACTG 2331 TATAGTTTTT TTTTTGTACA TATAATGATT AAACGGTCAT CCAACATCTC GTTGACAGAT 2391 CTCTCAGTAC GCGAAATCCC TGACTATCAA AGCAAGAACC GATGAAGAAA AAAACAACAG 2451 TAACCCAAAC ACCACAACAA ACACTTTATC TTCTCCCCCC CAACACCAAT CATCAAAGAG 2511 ATGTCGGAAC ACAAACACCA AGAAGCAAAA ACTAACCCCA TATAAAAACA TCCTGGTAGA 2571 TAATGCTGGT AACCCGCTCT CCTTCCATAT TCTGGGCTAC TTCACGAAGT CTGACCGGTC 2631 TCAGTTGATC AACATGATCC TCGAAATGGG TGGCAAGCAT CGTTCCAGAC CTGCCTCCTC 2691 TGGTAGATGG AGTGTTGTTT TTGACAGGGG ATTACAAGTC TATTGATGAA GATACCCTAA 2751 AGCAACTGGG GGACGTTCCA ATATACAGAG ACTCCTTCAT CTACCAGTGT TTTGTGCACA 2811 AGACATCTCT TCCCATTGAC ACTTTCCGAA TTGACAAGAA CGTCGAC 2858

[Brief description of drawings]

FIG. 1 is a diagram showing a model of functional sharing of Asn-linked sugar chains in erythropoietin. In the figure, Man means mannose, GlcNAc means N-acetylglucosamine, and Fuc means fucose.

FIG. 2 is a diagram showing a sugar chain structure model of a glycoprotein in baker's yeast. In the figure, M is mannose, 2 is α-1,2.
Bond 3, 3 means α-1,3 bond, 6 means α-1,6 bond and 4 means β-1,4 bond. Also, N-linke
“Ma” in the d sugar chain means a mannose sugar synthesized in the endoplasmic reticulum (ER).

FIG. 3 is a view showing a plasmid pKM049 in which a baker's yeast-derived OCH1 gene is subcloned.

FIG. 4 shows a gene fragment inserted into pKM50 (P. pastoris chromosome DN having homology with baker's yeast OCH1 gene).
A fragment map of (A fragment) is shown. The hatched region indicates the OCH1 gene translation region predicted from the determined nucleotide sequence.

FIG. 5 is a diagram showing the homology between the amino acid sequence encoded by the baker's yeast OCH1 gene (upper column) and the amino acid sequence encoded by P. pastoris sugar chain-extended DNA (lower column). □
Indicates an asparagine sugar chain addition site.

FIG. 6: OCH1 protein (A) derived from baker's yeast and P.pa
Hydrophobicit of storis-derived sugar chain elongation protein (B)
It is the figure which compared the y profile.

FIG. 7 is a photograph instead of a drawing, which shows the result of Genomic Southern Hybridization analysis using a sugar chain-extended DNA of Pichia yeast as a probe.

FIG. 8: P. pastoris sugar chain extension DNA disruption plasmid (p
It is a figure which shows the restriction enzyme map of KM74).

FIG. 9 is a diagram showing the structures in the vicinity of the sugar chain extension gene loci of the chromosomal DNAs of the sugar chain extension DNA-disrupted strain KM74-2 and the wild strains GTS115 and KM45, in which the underline in the figure indicates GenomicS.
It is the figure which showed the position of the probe used for the outhern Hybridization analysis. In the figure, E is EcoRI and Bg
Means BglII.

FIG. 10 is a photograph instead of a drawing, showing the results of Genomic Southern Hybridization analysis using the probe 1 shown in FIG. 9 for the sugar chain-extended DNA-disrupted strain KM74-2 and wild-type strains GTS115 and KM45.

FIG. 11 is a photograph instead of a drawing, which shows the results of Genomic Southern Hybridization analysis using the probe 2 shown in FIG. 9 for the sugar chain-extended DNA-disrupted strain KM74-2 and the wild-type strains GTS115 and KM45.

FIG. 12 is a photograph instead of a drawing, showing an electrophoretic image of SDS-PAGE analysis of the sFcεRIα chain protein produced by the P. pastoris sugar chain-expanded DNA-disrupted strain. 1:
sFcεRIα (KM45), 2: sFcεRIα (K
M74-2), 3: sFcεRIα (KM74-5),
4: PNGaseF-treated KM45-sFcεRIα,
5: PNGaseF treated KM74-2-sFcεRI
α, 6: PNGaseF-treated KM74-5-sFcεR
Iα

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location C12N 9/99 C12N 9/99 C12P 21/02 C12P 21/02 C // (C12P 21/02 C12R 1:84)

Claims (6)

[Claims] 1. A part of DNA having a base sequence encoding a protein involved in sugar chain elongation of the glycoprotein of the following (A) is modified so that the production of a functional product encoded by the DNA is at least suppressed. By culturing a modified Pichia yeast strain having the ability to extend the sugar chain of a glycoprotein as compared to a natural Pichia yeast strain by having the DNA thus obtained, and collecting the glycoprotein from the culture. A method for producing a characteristic glycoprotein. (A) A protein derived from a yeast of the genus Pichia, which has the amino acid sequence shown below substantially in the N-terminal region. Embedded image 2. The method for producing a glycoprotein according to claim 1, wherein the protein involved in sugar chain elongation of the glycoprotein has substantially the amino acid sequence shown below. Embedded image 3. The method for producing a glycoprotein according to claim 1, wherein the DNA having a base sequence encoding a protein involved in sugar chain elongation of glycoprotein has a base sequence shown below. Embedded image 4. An embodiment of modification of DNA modified so that production of a functional product is at least suppressed is insertion of a transformation marker gene into a nucleotide sequence of a DNA encoding a protein involved in sugar chain elongation. The method for producing the glycoprotein according to claim 1. 5. The transformation marker gene is selected from the group consisting of a baker's yeast-derived SUC2 gene, a Pichia yeast-derived HIS4 gene, an ARG4 gene, a URA3 gene and a G418 resistance gene. Item 4. A method for producing a glycoprotein according to Item 4. 6. The glycoprotein is selected from the group consisting of soluble high affinity IgE receptor α chain (sFcεRIα), chymase, pro-urokinase-annexin V fusion protein, urinary trypsin inhibitor, IGF1 binding protein 3 (IGF1BP3). It is any one, The manufacturing method of the glycoprotein in any one of Claims 1-5.