JP2001161376A - GLYCOSYLTRANSFERASE GENE och1 DERIVED FROM FISSION YEAST - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for specifying a gene encoding a glycosyltransferase derived from a fission yeast, and a method for producing an extraneous protein having less added sugar chains using a fission yeast in which the function of the gene was lost. SOLUTION: The finding that och1+ having a specific base sequence derived from a fission yeast Schizosaccharomyces pombe is homologous to a glycosyltransferase of a budding yeast permits producing a protein in which a high mannose-type sugar chain was not added using a fission yeast in which the function of the gene was lost, i.e., och+-destroyed strain.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a DNA encoding a glycosyltransferase of fission yeast, and a method for producing a fission yeast having a reduced ability to add a sugar chain to a protein by deficient function of the DNA. And a method for producing a foreign protein with little sugar chain addition using the fission yeast.

[0002]

2. Description of the Related Art Yeasts have a higher division rate than animal cells and can grow on inexpensive media, and have similar post-translational modifications to animal cells as compared to bacteria. Since sugar chains that do not add in bacteria are added, recombinant DN
It attracted attention as a host for protein production using DNA obtained by A technology. However, some glycosyltransferases are different between animal cells and yeast. Therefore, proteins produced in yeast contain a large amount of mannose glycan, which is not found in animal cells (hereinafter referred to as high-mannose type sugar chains). Many cases have been added. The addition of a high-mannose type sugar chain has an effect on protein productivity, protein antigenicity, pharmacokinetics, and the like, and has been a major problem in recombinant protein production in yeast.

[0003] Yeasts are broadly classified into budding yeasts and fission yeasts. In budding yeasts, a glycosyltransferase gene OCH1 that adds a high-mannose type sugar chain has been found (Nagasu, T., et.a.
l., Yeast, 8, 535-547, 1992), it was found that when a yeast strain lacking OCH1 was used to produce a recombinant protein, a high-mannose-type sugar chain was not added. However, in fission yeast, homologs to OCH1 of budding yeast were not found by ordinary methods such as hybridization, and mutants to which high-mannose-type sugar chains were not added could not be produced.

[0004]

An object of the present invention is to specify a gene encoding a glycosyltransferase of fission yeast, and to produce a protein with little sugar chain addition using the fission yeast having lost the function of the gene. To make it happen.

[0005]

Means for Solving the Problems The present inventors can introduce a genomic DNA library of fission yeast into a fission yeast mutant that can grow only under conditions of adjusted osmotic pressure at high temperatures, and complement its properties. From the genomic DNA fragment, a gene consisting of the nucleotide sequence of SEQ ID NO: 1 having homology to OCH1 of budding yeast was found. The gene is thought to be a gene encoding glycosyltransferase of fission yeast, and when the gene was deleted from fission yeast, the high-mannose type sugar chain that was added in the fission yeast that was not deleted was added. The inventor has found that it has disappeared, and has completed the present invention.

That is, the present invention provides the following (a) to (c):
The DNA described in any of the above (hereinafter referred to as “och1⁺"
). (a) a protein consisting of the amino acid sequence of SEQ ID NO: 2
DNA to encode.  (b) DNA comprising the nucleotide sequence of SEQ ID NO: 1. (c) a DNA comprising the nucleotide sequence of SEQ ID NO: 1 and a string
Hybridizes under gentle conditions and has glycosyltransferase activity
Fission yeast-derived DNA encoding a protein having
Och1⁺(Hereinafter referred to as “Och1” protein)
About).

Here, one example of the stringent condition is as follows.
Hybridization in 4 x SSC at 65 ° C, then
Wash in 0.1 × SSC at 65 ° C. for 1 hour. Alternatively, stringent conditions may include 42% in 50% formamide.
° C 4 x SSC.

[0008] The present invention also relates to a method for losing the function of och1 ^{+ in} a fission yeast gene using och1 ⁺ or a part thereof.

[0009] Here, to "delete the function of och1 ⁺ ", the region encoding the och1 ⁺ protein is mutated to
Includes both loss of function of one protein and reduction of och1 ⁺ expression. “Let the function of the Och1 protein be lost” means that the Och1 protein has been modified so that an excess of high-mannose-type sugar chains is not added to most of the proteins produced by the fission yeast. In addition, "to reduce the expression of och1 ^+", och1 in the genome of the fission yeast ⁺
Or by introducing a mutation into its expression control region,
This refers to reducing the amount of Och1 protein produced by the fission yeast compared to the amount of Och1 protein produced by the fission yeast not introducing the mutation.

Further, the present invention provides a fission yeast (hereinafter referred to as an och1 ⁺ disrupted strain) having the ability to produce a protein with reduced glycosylation by deficient och1 ⁺ function, a method for producing the same, and a method for producing the same. A method for producing a fission yeast which can be produced, preferably Schizosaccharomyces pombe, the fission yeast (och1 ⁺ disrupted strain) to produce a protein with little glycosylation,
And a protein with a small amount of glycosylation produced by the method.

[0011]

Embodiments of the present invention will be described below in detail.

[0012] DNA containing och1 ⁺ or a part thereof is prepared by setting primers based on the nucleotide sequence of SEQ ID NO: 1 and performing PCR using fission yeast genomic DNA or cDNA as a template, or fission yeast RNA. It can be obtained by performing RT-PCR on a template. Alternatively, a probe is synthesized based on the nucleotide sequence shown in SEQ ID NO: 1, a clone that hybridizes with the probe is selected from a genomic DNA library or cDNA library of fission yeast, and the nucleotide sequence is determined. Then, a clone containing och1 ⁺ or a part thereof may be selected.

Furthermore, och1 which affects the expression of Och1 protein
^{The +} expression control region synthesizes a probe based on the nucleotide sequence of SEQ ID NO: 1, selects a clone that hybridizes with the probe from the fission yeast genomic DNA library, determines the nucleotide sequence, and determines the och1 ⁺ Can be obtained by selecting a clone containing an upstream or downstream sequence. A probe may be synthesized based on the determined upstream or downstream base sequence, and a clone further containing the upstream or downstream sequence may be selected.

[0014] The region having the expression regulating activity is usually present within 1 kbp upstream and 500 bp downstream of the coding region. In fission yeast in which the region isolated by homologous recombination has been deleted, the amount of och1 ⁺ mRNA quantified by Northern blotting is reduced, or excessive mannose-type sugar chains are added to the protein produced by the fission yeast By analyzing whether or not there is no expression, it can be determined whether or not the isolated region has an expression controlling activity.

The obtained och1 ⁺ gene can be inserted into an expression vector to produce an Oct1 protein. The expression vector is inserted into, for example, pGEX when the host is Escherichia coli, for example, pcL, pREP when the host is yeast, or pCMV, pSRα, for example, when the host is a mammalian cell. Insertions are described, for example, in Sambruck, J., Fritsch, EF, and Maniatis, T.
(1989) Molecular Cloning: A Laboratory Manual, Col
d Spring Harbor Laboratory, Cold Spring Harbor, NY
Can be performed according to the method described in (1). It is desirable that the Oct1 protein be expressed as a fusion protein with His6 and GST, since subsequent purification will be easy.

The expression vector into which the och1 ⁺ gene has been inserted is
It is introduced into the host in a manner appropriate for the host. The introduction, for example,
Sambruck, J., Fritsch, EF, and Maniatis, T. (19
89) Molecular Cloning: A Laboratory Manual, Cold Sp
This can be performed according to the method described in Ring Harbor Laboratory, Cold Spring Harbor, NY.

After culturing the host into which the och1 ⁺ gene has been introduced under appropriate conditions, the host is disrupted and the protein fraction is extracted.
Crushing can be performed using glass beads or ultrasonic waves when the host is Escherichia coli or yeast, and using a homogenizer when the host is mammalian cells. When a device is devised so that the protein produced in the expression vector is secreted into the culture supernatant, the target protein can be recovered from the culture supernatant.

From the extracted protein fraction, electrophoresis, various chromatography, for example, affinity column,
The och1 ⁺ gene product can be purified by a standard method using an ion exchange column, a gel filtration column, or the like. When the introduced gene is designed to encode the fusion protein, for example, GST can be easily purified using a glutathione column, and His6 can be easily purified using a nickel sepharose column.

The method of mutating the och1 ⁺ protein-coding region involves homologous recombination between och1 ⁺ in the fission yeast gene and a circular or linear DNA containing the mutated och1 ⁺ or a part thereof. Och1 ⁺ in the fission yeast gene
Is basically replaced with och1 ⁺ into which a mutation has been introduced. The DNA containing a part of och1 ⁺ used for recombination is usually 500 bp or more, and preferably 1 kbp or more.

Och obtained by PCR or cloning
Methods for introducing mutations into DNA containing ¹⁺ or a part thereof include recombinant DNA technology (Sam.
bruck, J., Fritsch, EF, and Maniatis, T. (1989)
Molecular Cloning: A Laboratory Manual, Cold Spri
ng Harbor Laboratory, Cold Spring Harbor, NY), P
Technology using CR (Ling MM. And Robinson BH., Anal.
Biochem. 254 (2): 157-78, 1997). For example, after cutting with an appropriate restriction enzyme,
An unrelated gene, preferably a selection marker gene capable of selecting a fission yeast that has undergone homologous recombination, and more preferably a method of inserting a uracil synthesis gene ura4 ⁺ as a selection marker can be mentioned. The method is not limited to this method as long as it can be performed. When there is no appropriate restriction enzyme site, an appropriate restriction enzyme site may be inserted by PCR or the like.

Circular or linear DNA containing och1 ⁺ or a part thereof into which a mutation has been introduced is introduced into fission yeast by a method such as a spheroplast method, a lithium acetate method, or an electroporation method. If the introduced mutant gene contains a selection marker, for example, ura4 ⁺ , culture conditions under which only fission yeast having the selection marker survive, for example, if the selection marker
If ura4 ⁺ , by culturing in a medium without uracil, homologous recombination occurred, and och1 ⁺
Och1 ⁺ disrupted strains can be selected.

A method for reducing the expression of the Och1 protein is to replace the DNA containing the region encoding the och1 ⁺ protein with
By using the cloned och1 ⁺ expression control region (including upstream or downstream sequences) and introducing mutations into the nucleotide sequence that affects Och1 protein expression, the aforementioned och1 ⁺
Can be performed in the same manner as in the case of the region encoding the protein.

As a method for decreasing the expression of Och1 protein, an expression vector into which a DNA encoding an RNA (antisense RNA) complementary to the och1 ⁺ transcript has been inserted into fission yeast is introduced. Methods such as reducing the expression of the protein are also conceivable. Here, “complementary” is not limited to completely complementary as long as expression of och1 ⁺ can be suppressed. Usually, it is sufficient that the complementarity is 80% or more, preferably 90% or more, and more preferably 95% or more.

The DNA encoding the antisense RNA usually needs to have a 20 bp chain length complementary to a site that plays an important role in the function of mRNA, such as a translation initiation site. However, in consideration of the fact that a higher-order structure prevents the antisense function, the longer chain length, preferably a chain length of 100 bp, can be used.

The fission yeast according to the present invention may be any one of the fission yeasts which have been subjected to a treatment for reducing the expression of the Och1 protein, and as a result, an excess of high-mannose-type sugar chains are not added to most of the protein to be produced. include.

It is determined whether an excessively high mannose type sugar chain is added. For example, since a high mannose type sugar chain shows diversity, for example, a protein to which a high mannose type sugar chain is added has various molecular weights. It can be estimated by showing. In other words, in fission yeast with wild-type och1 ⁺ , if a protein showing a smearing pattern by electrophoresis shows a sharp pattern in fission yeast with mutation, there is no excess high mannose type sugar chain added It can be inferred that the protein was produced.

Hereinafter, a specific example of a method for losing the function of och1 ⁺ will be described. Linear D containing och1 ⁺ with loss of function
NA as, for example och1 ⁺ linear DNA that about 60% central and replaced with uracil synthesis system gene ura4 ⁺ coding region (och1 :: ura
4 ⁺ ). By introducing it into fission yeast lacking ura4 ^+, to cause homologous recombination between the och1 ⁺ in fission yeast and och1 :: ura4 ^+. After that, the cells were cultured in a medium without uracil and had ura4 ⁺ , that is, o
By ch1 ⁺ to select the fission yeast replacing a och1 :: ura4 ^+, can be obtained fission yeast was lost the function of the och1 ^+, i.e. och1 ⁺ destruction strain.

[0028] The present invention further relates to a method for producing a protein with less glycosylation using an och1 ⁺ disrupted strain. Here, the protein may be any protein as long as it is a protein produced by fission yeast, and may be a protein encoded by any of genes derived from fission yeast and foreign genes not derived from fission yeast. Here, the exogenous gene is a gene that is desired to be expressed in fission yeast, and may be a gene derived from any organism such as an animal, a yeast, or a bacterium, but is preferably an animal-derived gene. The foreign gene may have an artificial sequence such as a fusion protein.

Examples of a method for introducing a gene include a spheroplast method, a lithium acetate method, and an electroporation method, but the present invention is not limited thereto. Specifically, for example, in the lithium acetate method, fission yeast in the logarithmic growth phase is incubated in lithium acetate at 30 ° C. for 1 hour, and then the introduced DNA and polyethylene glycol solution are added, followed by incubation at 30 ° C. for 1 hour. This was heat-treated at 43 ° C for 15 minutes, and then the cells were collected by centrifugation and suspended in a medium.
Incubate with shaking at 2 ° C for 2 hours. When this culture solution is spread on a selective agar medium and cultured at 30 ° C. for 3 days, only yeast cells into which a gene has been introduced and which can grow on the selective medium form colonies.

The present invention also includes a protein with a small amount of glycosylation produced by the och1 ⁺ disrupted strain. Here, the protein produced from the fission yeast according to the present invention is:
It can be purified from the culture supernatant or cells by standard methods such as affinity column, ion exchange column and gel filtration column. When the introduced gene is designed to encode a fusion protein, for example,
In the case of a fusion protein with GST, it is preferable to purify it with an affinity column with the fusion protein, and in the case of GST, it is preferable to purify it with a glutathione column.

It should be noted that the operations described above are performed in appropriate manuals, such as Sambruck, J., Fritsch, EF, and Maniati.
s, T. (1989) Molecular Cloning: A Laboratory Manua
l, Cold Spring Harbor Laboratory, Cold Spring Harbo
r, NY, etc.

[0032]

EXAMPLES The present invention will be described in more detail with reference to the following Examples, but it should not be construed that the invention is limited thereto.

Example 1 Homology of Fission Yeast och1 ⁺ and Saccharomyces cerevisiae OCH1 Consisting of SEQ ID NO: 1 Osmotic pressure of a fission yeast mutant cloned from a fission yeast genomic DNA library at high temperatures Genomic DNA that can complement the ability to grow only under conditions that prepare
From the fragment, the fission yeast gene och1 ⁺ consisting of the nucleotide sequence of SEQ ID NO: 1 was found. Then, as shown in FIG. 1, fission yeast och1 ⁺ (SpOCH1) and budding yeast OCH1 (ScOCH1)
Homology was found between 1).

[Example 2] Preparation of och1 ⁺ disrupted strain DNA containing the mutation-introduced nucleotide sequence of SEQ ID NO: 1
Causes homologous recombination between the fission yeast och1 ⁺ and och1 ⁺
A disrupted strain was prepared. Contains the nucleotide sequence of SEQ ID NO: 1
DNA in which the region flanked by two HindIII cleavage sites present in the och1 ⁺ translation region of DNA is replaced with fission yeast ura4 ⁺
(FIG. 2). Then, the fission yeast was transformed by the spheroplast method, and cultured in a selective medium not containing uracil to have ura4 ⁺ , that is, the mutation was introduced.
Fission yeast having ch1 ⁺ was selected and och1 ⁺ disrupted strain was prepared.

[0035] in [Example 3] och1 ⁺ form microscopic fission yeast deleted, compared with wild-type fission yeast and fission yeast was deleted och1 ^+. As shown in FIG. 3, the fission yeast from which och1 ⁺ has been deleted has a rounder and shorter form than the wild-type fission yeast. In addition, they tend to aggregate and have a slow growth rate. 37 ℃
Although it shows temperature sensitivity that it can not grow in
When about 1.5% sorbitol is added and high osmotic pressure is applied,
It can grow even at 37 ° C.

Example 4 Cultivation of och1 ⁺ Disrupted Strain and Analysis of Produced Protein Whether or not disruption of och1 ⁺ had an effect on glycosylation of the produced protein was examined. The och1 ⁺ disrupted strain and the wild-type fission yeast were cultured, and the produced glycoprotein, here, acid phosphatase was electrophoresed, and the activity was stained to examine the molecular weight distribution of acid phosphatase. As a result, as shown in FIG. 4, the acid phosphatase showed a smear pattern with a large molecular weight in the wild-type strain (lane 1), whereas a sharp band with a small molecular weight in the och1 ⁺ disrupted strain (lane 2) ). This suggests that high-mannose-type glycosylation with high diversity and high molecular weight occurs in the wild-type strain and not in the och1 ⁺ disrupted strain.

Further, in order to examine whether such a difference in electrophoretic mobility was due to the addition of a sugar chain, a sugar chain cleavage treatment with endoglycosidase H (Endo H) was performed. Since endoglycosidase H is an enzyme that cuts between two N-acetylglucosamine residues at the base of an N-linked sugar chain, the glycoprotein treated with endoglycosidase H has a molecular weight estimated from the polypeptide portion. Shows almost the same mobility. As a result, the bands of acid phosphatase derived from the wild type and the och1 ⁺ disrupted strain in lanes 3 and 4 treated with endoglycosidase H appeared in the same place, and the difference in mobility between lanes 1 and 2 was due to the N-linked This was shown to be due to the difference in the size of the sugar chain.

[0038] [Example 5] och1 ⁺ mannosyltransferase activity mannosyltransferase activity och1 ⁺ gene product of a gene product, the Man9GlcNAc2-PA and GDP- mannose fluorescently labeled as a substrate,
och1 ⁺ disrupted strain, och1 ⁺ och1 ⁺ disrupted strain was introduced expression vector, and the crude extract was disrupted wild strain was measured as the enzyme. The fission yeast och1 ⁺ gene was replaced with pREP1 (Maundrell,
K. 1993. Thiamine-repressible expression vectors p
REP and pRIP for fission yeast.Gene 123, 127-13
0) It was incorporated into an expression vector to prepare an och1 ⁺ expression vector. och1 ⁺ crushed och1 ⁺ disrupted strain overexpressing the introduced och1 ⁺ gene product expression vectors, och1 ⁺ disrupted strain was introduced only vector as a control, and the cells of the wild strain, which was precipitated with 100,000 xg Was used as the enzyme. 50 mM Tris HCl (pH 7.5), 10 mM MnCl ₂ , 0.6% T
riton X-100, 0.5 mM 1-deoxymannojirimycin, 2 mM PA
-sugar chain, 1 mM GDP-mannose was used as a reaction solution, and an enzyme was added to start an enzyme reaction. After reacting at 30 ° C for 10, 20, 30 minutes, the reaction is stopped by treating at 99 ° C for 5 minutes, and the reaction product is removed.
Separation by HPLC using a TSK Gel Amide-80 column,
The enzymatic reaction was analyzed.

As shown in FIG. 5, Man9Glc was found in the och1 ⁺ disrupted strain.
No transfer of mannose to NAc2-PA was observed,
Mannose translocation was observed in the wild strain, and more mannose translocation was observed in the och1 ⁺ disrupted strain into which the och1 ⁺ expression vector was introduced and the och1 ⁺ gene product was overexpressed. From these results, it was confirmed that the och1 ⁺ gene product had an activity as a mannose transferase activity and was involved in the same reaction as the S. cerevisiae OCH1 gene product.

[0040]

Industrial Applicability According to the present invention, fission yeast in which the function of glycosyltransferase has been lost can be obtained, and it has become possible for fission yeast to produce a protein with less added sugar chains.

[0041]

[Sequence List] SEQUENCE LISTING <110> Eisai Co., Ltd. AGENCY OF INDUSTRIAL SCIENCE AND TECHNOLOGY <120> glycosyltransferase gene och1 ⁺ <130> E1-102DP1 <150> JP 1999-246494 <151> 1999-08-31 <160> 2 <170> PatentIn Ver. 2.0 <210> 1 <211> 1191 <212> DNA <213> Schizosaccharomyces pombe <220><221> CDS <222> (1) .. (1191) <400> 1 atg ttg aga ctc cga ttg aga agt att gta ata gga gct gcg ata gcg 48 Met Leu Arg Leu Arg Leu Arg Ser Ile Val Ile Gly Ala Ala Ila Ala 1 5 10 15 gga tcc ata ctg ttg tta ttc aat cat ggt agc ata atg gaa 96 Gly Ser Ile Leu Leu Leu Phe Asn His Gly Ser Ile Glu Gly Met Glu 20 25 30 gat ttg aca gag att tcg atg ttg gag gat tac act ccg gaa gcc gct 144 Asp Leu Thr Glu Ile Ser Met Leu Glu Asp Tyr Thr Pro Glu Ala Ala 35 40 45 aat aaa gat tat gtt ggt caa cag gag gag gag gaa ctt ttg tat gat 192 Asn Lys Asp Tyr Val Gly Gln Gln Glu Glu Glu Glu Leu Leu Tyr Asp 50 55 60 caa ccg tct tac att gaa gaa gaa gaa gat cca gat ttg gaa gct tac 240 Gln Pro Ser Tyr Ile Glu Glu Glu Glu Asp Pro Asp Leu Glu Ala Tyr 65 70 75 80 ttg agc gat ttg gag agg gaa gag ctg gaa cac agc ttg gaa gaa ctt 288 Leu Ser Asp Leu Glu Arg Glu Glu Leu Glu His Ser Leu Glu Glu Leu 85 90 95 agat gaaga tat aaa ctt cat cta cgg tac tcc ttt tca cag 336 Asp Glu Glu Asn Asn Tyr Lys Leu His Leu Arg Tyr Ser Phe Ser Gln 100 105 110 ctt caa gat ttt gac gaa gaa aat gaa gct gta cac atg atc gtt cct 384 Leu Gln Asp Phe Asp Glu Glu Asn Glu Ala Val His Met Ile Val Pro 115 120 125 aaa gat act tat gaa ttt gag gtg cct tat cac gct gac att ccc aag 432 Lys Asp Thr Tyr Glu Phe Glu Val Pro Tyr His Ala Asp Ile Pro Lys 130 135 140 tta ata tgg caa act tcc aag gac cct ttt gat aga gag gtt atg aag 480 Leu Ile Trp Gln Thr Ser Lys Asp Pro Phe Asp Arg Glu Val Met Lys 145 150 155 160 tac act cgg ttt tgg aga atc aac cat ccc agt tat tct cat gct gtt 528 Tyr Thr Arg Phe Trp Arg Ile Asn His Pro Ser Tyr Ser His Ala Val 165 170 175 tta gac gat gag cag tct aaa aaa gca ttg gtc atc agt agc ttt ggc gat 576 Leu Asp Asp Glu Gln Ser Lys Ala Le u Val Ile Ser Ser Phe Gly Asp 180 185 190 tca tca gtt tcc aag att tca caa gcg tat gca atg atg cct ctg cct 624 Ser Ser Val Ser Lys Ile Ser Gln Ala Tyr Ala Met Met Pro Leu Pro 195 200 205 gtt ctg aag gcc gat ttc ttt cgg tat cta gtg tta ttg gca aaa ggt 672 Val Leu Lys Ala Asp Phe Phe Arg Tyr Leu Val Leu Leu Ala Lys Gly 210 215 220 ggt att tat agc gac att gat acg gca cca ttg aag cat ata aac at Gly Ile Tyr Ser Asp Ile Asp Thr Ala Pro Leu Lys His Ile Asn Asn 225 230 235 240 tgg atc cct cgt gaa tat cgt aag cgt aat att cga ctg atc gtt ggc 768 Trp Ile Pro Arg Glu Tyr Arg Lys Arg Asn Ile Arg Leu Ile Val Gly 245 250 255 att gaa gca gat ccc gac cgc cct gat tgg aac gac tac tat gcc agg 816 Ile Glu Ala Asp Pro Asp Arg Pro Asp Trp Asn Asp Tyr Tyr Ala Arg 260 265 270 cgt gta caa ttc tgt cag tgg acc ata gct gct gca cca ggc cat cca 864 Arg Val Gln Phe Cys Gln Trp Thr Ile Ala Ala Ala Pro Gly His Pro 275 280 285 att ctt tgg gaa ctt gtt cgt aga att acc gat gaa act tgg aag ctg 912 Ile Leu Trp Glu Leu Va l Arg Arg Ile Thr Asp Glu Thr Trp Lys Leu 290 295 300 cat gat tca aag aag ctt tcg aaa aat ggc gag tca gta atg gag tgg 960 His Asp Ser Lys Lys Leu Ser Lys Asn Gly Glu Ser Val Met Glu Trp 305 310 315 320 act ggt cca ggt att tgg acc gat gcc att atg gat tat tta aat tgg 1008 Thr Gly Pro Gly Ile Trp Thr Asp Ala Ile Met Asp Tyr Leu Asn Trp 325 330 335 cag tat ggc cct ttc tcg gtt gag aat atc acc aat tta gaa gaa cct 1056 Gln Tyr Gly Pro Phe Ser Val Glu Asn Ile Thr Asn Leu Glu Glu Pro 340 345 350 tat ctt gtt gga gat gtt tta atc cta ccc ata act gca ttt agt cct 1104 Tyr Leu Val Gly Asp Val Leu Ile Leu Pro Ile Thr Ala Phe Ser Pro 355 360 365 ggt gtc ggt cac atg ggt agc aag tct cca aat gat ccg atg gcg tat 1152 Gly Val Gly His Met Gly Ser Lys Ser Pro Asn Asp Pro Met Ala Tyr 370 375 380 gtt caa cat ttc ttt gcc ggt tca tgg aaa gat gat tga 1191 Val Gln His Phe Phe Ala Gly Ser Trp Lys Asp Asp 385 390 395 <210> 2 <211> 396 <212> PRT <213> Schizosaccharomyces pombe <400> 2 Met Leu Arg Leu Arg Leu Arg Ser I le Val Ile Gly Ala Ala Ile Ala 1 5 10 15 Gly Ser Ile Leu Leu Leu Phe Asn His Gly Ser Ile Glu Gly Met Glu 20 25 30 Asp Leu Thr Glu Ile Ser Met Leu Glu Asp Tyr Thr Pro Glu Ala Ala 35 40 45 Asn Lys Asp Tyr Val Gly Gln Gln Glu Glu Glu Glu Leu Leu Tyr Asp 50 55 60 Gln Pro Ser Tyr Ile Glu Glu Glu Glu Asp Pro Asp Leu Glu Ala Tyr 65 70 75 80 Leu Ser Asp Leu Glu Arg Glu Glu Leu Glu His Ser Leu Glu Glu Leu 85 90 95 Asp Glu Glu Asn Asn Tyr Lys Leu His Leu Arg Tyr Ser Phe Ser Gln 100 105 110 Leu Gln Asp Phe Asp Glu Glu Asn Glu Ala Val His Met Ile Val Pro 115 120 125 Lys Asp Thr Tyr Glu Phe Glu Val Pro Tyr His Ala Asp Ile Pro Lys 130 135 140 Leu Ile Trp Gln Thr Ser Lys Asp Pro Phe Asp Arg Glu Val Met Lys 145 150 155 160 Tyr Thr Arg Phe Trp Arg Ile Asn His Pro Ser Tyr Ser His Ala Val 165 170 175 Leu Asp Asp Glu Gln Ser Lys Ala Leu Val Ile Ser Ser Phe Gly Asp 180 185 190 Ser Ser Val Ser Lys Ile Ser Gln Ala Tyr Ala Met Met Pro Leu Pro 195 200 205 Val Leu Lys Ala Asp Phe Phe Arg Tyr Leu Val Leu Leu Ala Lys Gly 210 215 220 Gly Ile Tyr Ser Asp Ile Asp Thr Ala Pro Leu Lys His Ile Asn Asn 225 230 235 240 Trp Ile Pro Arg Glu Tyr Arg Lys Arg Asn Ile Arg Leu Ile Val Gly 245 250 255 255 Ile Glu Ala Asp Pro Asp Arg Pro Asp Trp Asn Asp Tyr Tyr Ala Arg 260 265 270 Arg Val Gln Phe Cys Gln Trp Thr Ile Ala Ala Ala Pro Gly His Pro 275 280 285 Ile Leu Trp Glu Leu Val Arg Arg Ile Thr Asp Glu Thr Trp Lys Leu 290 295 300 His Asp Ser Lys Lys Leu Ser Lys Asn Gly Glu Ser Val Met Glu Trp 305 310 315 320 Thr Gly Pro Gly Ile Trp Thr Asp Ala Ile Met Asp Tyr Leu Asn Trp 325 330 335 Gln Tyr Gly Pro Phe Ser Val Glu Asn Ile Thr Asn Leu Glu Glu Pro 340 345 350 Tyr Leu Val Gly Asp Val Leu Ile Leu Pro Ile Thr Ala Phe Ser Pro 355 360 365 Gly Val Gly His Met Gly Ser Lys Ser Pro Asn Asp Pro Met Ala Tyr 370 375 380 val Gln His Phe Phe Ala Gly Ser Trp Lys Asp Asp 385 390 395

[Brief description of the drawings]

FIG. 1 is a diagram showing the homology of fission yeast och1 ⁺ and budding yeast OCH1.

2 is a diagram showing the inserted construct of DNA the och1 ⁺ center of ura4 ^+.

FIG. 3 is a photograph showing the form of fission yeast in which och1 ⁺ has been deleted.

FIG. 4 is a photograph showing an analysis result of acid phosphatase produced in an och1 ⁺ disrupted strain.

FIG. 5 shows the mannose transferase activity of the och1 ⁺ gene product.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI theme coat ゛ (Reference) C12N 1/19 C12N 1/21 1/21 9/10 5/10 C12P 21/02 C 9/10 C12R 1 : 645) C12P 21/02 (C12N 1/00 U // (C12N 15/09 ZNA C12R 1: 645) C12R 1: 645) (C12N 9/10 (C12N 1/00 C12R 1: 645) C12R 1: 645 ) (C12P 21/02 C (C12N 9/10 C12R 1: 645) C12R 1: 645) C12N 15/00 ZNAA (C12P 21/02 5/00 A C12R 1: 645) C12R 1: 645) (72) Invention Person Tatsuo Watanabe 4-14-9 Kinoshitahigashi, Inzai City, Chiba Prefecture (72) Inventor Takehiko Yokoo 1-3-1 Higashi, Tsukuba City, Ibaraki Prefecture Within the Institute of Biotechnology, Industrial Technology Institute (72) Inventor Yoshifumi Jigami Tsukuba, Ibaraki Prefecture 1 city east No. 1-3 F-term in the Institute of Biotechnology, Industrial Technology Research Institute (Reference) 4B024 AA20 BA10 CA01 DA12 EA04 FA01 GA11 GA25 HA01 HA03 4B050 CC01 DD04 LL05 4B064 AG01 CA06 CA19 CC24 4B065 AA72X AA72Y AB01 AC14 CA24 CA29 4H045 DA8953 FA74

Claims (14)

[Claims] 1. A DN according to any one of the following (a) to (c):
DNA encoding a glycosyltransferase consisting of A. (a) DNA encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2. (b) DNA comprising the nucleotide sequence of SEQ ID NO: 1. (c) a fission yeast-derived DNA that hybridizes with a DNA consisting of the nucleotide sequence of SEQ ID NO: 1 under stringent conditions and encodes a protein having glycosyltransferase activity. 2. A vector into which the DNA according to claim 1 has been inserted. 3. A host cell into which the vector according to claim 2 has been introduced. A protein encoded by the DNA according to claim 1. 5. A method for producing a fission yeast having the ability to produce a protein with reduced glycosylation, wherein the function of the DNA according to claim 1 in the genome of the fission yeast is deleted. Method. 6. The DN of claim 1 in the fission yeast genome.
The method according to claim 5, wherein the function of the DNA is deleted by mutating the protein coding region of A. 7. The DN of claim 1 in a fission yeast genome.
The method according to claim 5, wherein the function of the DNA is lost by decreasing the expression of A. 8. The function of the DNA of claim 1 in the fission yeast genome by introducing the DNA of claim 1 or a part thereof into which a mutation has been introduced into the protein coding region and by homologous recombination with the DNA. 7. The method according to claim 6, wherein is deleted. 9. The DN according to claim 1, wherein the mutation has been introduced.
8. The method according to claim 7, wherein the function of the DNA according to claim 1 in the fission yeast genome is deleted by introducing DNA which is an expression control region of A or a part thereof and homologous recombination with the DNA. 10. The function of the DNA of claim 1 in the fission yeast genome is deleted by introducing and expressing a DNA encoding an RNA complementary to the transcription product of the DNA of claim 1. 7. The method according to 7. 11. The fission yeast genome according to claim 1, which is in the fission yeast genome.
A fission yeast capable of producing a protein having a reduced glycosylation due to a loss of DNA function. 12. A Schizosaccharomyces pombe,
The fission yeast according to claim 11. 13. A method for producing a protein with reduced glycosylation using the fission yeast according to claim 11 or 12. 14. A protein with reduced glycosylation produced by the method according to claim 13.