WO2004003205A1

WO2004003205A1 - Hansenula polymorpha mutant strains with defect in outer chain biosynthesis and the production of recombinant glycoproteins using the same strains

Info

Publication number: WO2004003205A1
Application number: PCT/KR2003/001285
Authority: WO
Inventors: Hyun-Ah Kang; Sang-Ki Rhee; Moo-Woong Kim
Original assignee: Korea Research Institute Of Bioscience And Biotechnology
Priority date: 2002-06-29
Filing date: 2003-06-30
Publication date: 2004-01-08
Also published as: AU2003246286A1; KR20040004089A

Abstract

The present invention relates to polynucleotide containing Hansunula polymorpha Hpoch 1 gene; polypeptide coded thereby; Hansunula polymorpha mutant wherein hyperglycosylation of glycoprotein id inhibited by the mutation of the Hansunula polymorpha HpOCH1, or Hansunula polymorpha natural mutant; recombinant Hansunula polymorpha strain expressing a foreign protein prepared by introducing a gene coding a foreign protein to the Hansunula polymorpha mutant or Hansunula polymorpha natural mutant; and a method for preparing a foreign protein comprising the steps of culturing said mutant under the condition that a foreign protein can be expressed, and isolating the foreign protein from the obtained culture broth.

Description

Hans enula polymorpha mutant strains with defect in outer chain biosynthesis and

the production of recombinant glycoproteins using the same strains

Field of the invention

The present invention relates to Hansenula polymorpha mutant strains

with a defect in the outer chain biosynthesis of glycoproteins and the production

method of recombinant glycoproteins using these strains. More specifically, the

present invention relates to the nucleic acid molecules containing H. polymorpha

HpOCHl gene, the polypeptides encoded by it, and H. polymorpha artificial

mutant strains or its natural mutant strains in which hyperglycosylation of

glycoproteins is prevented. Furthermore, the present invention relates to

recombinant H. polymorpha strains expressing a foreign protein produced by

transformation with a gene encoding a foreign protein, and the production method

of a foreign protein, which comprises cultivating the strains under conditions that

allow them to express the foreign protein and isolating the expressed foreign

protein from the cultures.

Background of the invention

In order to express a foreign protein recombinantly on a large scale, an

optimal expression system should be selected to establish an efficient production

system because amounts, solubility, locations and modifications etc. of expressed proteins are dependent on host cell lines or features of desired proteins. For

large-scale expression of proteins, various host systems including bacteria, yeasts,

fungi, plants and animals have been developed. Among them, microbes have

been wildly used to express recombinant proteins because of easy culture thus

getting a high concentration of recombinant protein with a low-cost.

Yeasts, microbes having features of the eukaryotic expression and

secretion of proteins, are a suitable expression system to produce recombinant

proteins of higher eukaryotes on a large-scale. In comparison to bacterial

expression systems, yeast expression systems have a major advantage in that, as

eukaryotic microbes, they have protein secretory organelles similar to those of

higher eukaryotes. Therefore, the secretory proteins in yeast become biologically

active through post-translational modifications such as digestion of secretory

signal sequences, formation of disulfide bonds, glycosylation etc. Furthermore,

the expressed recombinant proteins can be easily recovered and purified, since

most yeast cells secrete only a small fraction of the proteins to the outside.

Recently, methylotrophic yeasts such as Hansenula polymorpha, Pichia

pastoris and other non-conventional yeasts have been developed as alternative

hosts, because they are able to replace the inherent disadvantages of the

traditioanl yeast Saccharomyces cerevisiae as hosts for industrial production of

desired proteins. The disadvantages of S. cerevisiae include instability of

expression vectors in long-term fermentation, hyperglycosylation of

glycoproteins, and low productivity of the expressed proteins in comparison to bacterial expression system (Gellissen, Appl. Microbiol. Biotechnol. 54, 741,

(2000)).

Most proteins utilized for medical therapeutic purposes in humans are

glycoproteins, which are modified by attachment of oligosaccharides via covalent

bonds in a secretory pathway. An important issue in large-scale protein

production in the field of biotechnology is the production of recombinant proteins

modified by suitable glycosylation because the structures and classes of

carbohydrates attached to the glycoproteins can greatly affect folding, secretion,

stability, half-life in serum, and antibody inductivity of the proteins.

Wild type yeasts have some limits as an expression system. The

recombinant glycoproteins expressed in S. cerevisiae have showed

hypermannosylation resulting from adding over 40 mannose residues to the

proteins and α 1,3-linked terminal mannose, which serves as an antigen in the human body (Romanos et al., Yeast 8, 423-488, 1992). In contrast the

recombinant proteins expressed in methylotrophic yeasts, H. polymorpha and P.

pastoris, have been reported to contain the mannose outer chains that are shorter

than those expressed in S. cerevisiae although they are still more

hyperglycosylated than native proteins (Bretthauer and Castellino, Biotechnol.

Appl. Biochem. 30, 193-200, 1999; Kang et al, Yeast 14, 371-381, 1998). These

methylotrophic yeasts are preferred over the wild type S. cerevisiae as a host

system for medical therapeutic proteins because they do not produce the α 1,3- linked terminal mannose, which can evoke an immune response. The core oligosaccharide is an intermediate of the biosynthesis pathway,

which is found in all eukaryotes from yeasts to mammalian cells. However, the

outer chains attached to the intermediate are differentially biosynthesized based

on species of proteins, cells and animals. Researchers have actively pursued the

development of a useful host system to produce recombinant glycoproteins, which

closely resemble native proteins containing proper outer chains, by means of

selecting mutant strains with defects in outer chain biosynthesis using an artificial

mutant method or manipulating the gene related to the chain biosynthesis using

molecular biological techniques.

In wild type S. cerevisiae, several strategies such as [³H]mannose suicide

selection, sodium orthovanadate resistance and hygromycin B sensitivity are used

to select the defective mutants of N-linked oligosaccharides biosynthesis

(Herscovics and Orlean, FASEB 7, 540-550, 1999). Functional complementation

experiments using these mutants led to the cloning of the OCHl gene (Ngd29)

playing an important role in the outer chain initiation (Nakanishi-Shindo et al., J.

Biol. Chem. 268, 26338-26345, 1993), the MNN9 gene regulating the outer chain

elongation and the MNN1 gene involved in attachment of the 1,3-linked terminal mannose (Gopal and Ballou, Proc. Natl. Acad. Sci. USA 84, 8824, 1987).

Those genes were targeted to make defective mutants by mutagenesis, which were

then developed as a host cell to produce recombinant glycoproteins (Kniskern et

al, Vaccine 12, 1021-1025, 1994; US Patent no. 5,798,226; US Patent no. 5,135,854). Methylotrophic yeasts have recently been in the spotlight as a suitable host

for recombinant protein expression over S. cerevisiae. However, a defective

mutant of the N-linked oligosaccharide biosynthesis in methylotrophic yeasts has

not yet been reported.

The goal of this invention was to develop a mutant using H. polymorpha,

a methylotrophic yeast, which can produce recombinant glycoproteins that are

suitable for use in the human body. This mutant was obtained by selection of a

defective mutant in the glycosylation pathway or by mutation of the OCHl gene

involved in the process. This defective mutant prevents hyperglycosylation of the

outer chains and is a suitable host for recombinant glycoproteins attached with

proper outer chains, which closely resemble the native proteins.

Summary of the invention

In order to develop a defective mutant of H. polymorpha for production of

recombinant N-linked glycoproteins closely resembling those of human, we

developed a method for selection of a defective mutant of the oligosaccharide

chain biosynthesis. We used sensitivity of sodium orthovanadate to select a

defective mutant of H. polymorpha, which exhibits more resistance against it.

We also cloned the OCHl gene involved in initiation of the outer chain

biosynthesis. The gene was mutated to make an OCHl deletion mutant (Δochl)

strain. This mutant strain is a suitable host, which provides techniques to produce recombinant glycoproteins close to the structure of original proteins with proper

outer oligosaccharide chains.

Brief description of the drawings

Figure 1 shows the difference in the resistance of H. polymorpha strains,

against sodium orthovanadate.

Figure 2 shows the phenotype of the H. polymorpha mutant, DL42-15.

Yeast cells in a log phase were serially diluted 1 to 10, 5 μl was spotted onto the

YPD plate and the cells were cultured for 2 days. A, YPD media containing 4 mM

sodium orthovanadate; B, YDP media at 45 ^°C ; C, YPD media containing 0.3%

sodium deoxycholate; D, YDP media at 37 ^°C .

Figure 3 shows the sequences of DNA and predicted amino acid of H..

polymorpha OCHl gene cloned in this study.

Figure 4 shows amino acid sequence alignment of the Ochlp of H.

polymorpha with homologues of other yeast strains. The numbers in parentheses

represent homology of Ochlp from other yeast strains versus Ochlp of H.

polymorpha. ΗpOchlp; H. polymorpha Ochl protein; ScOchlp, S. cerevisiae

Ochl protein; ScΗoclp, S. cerevisiae Hocl protein; CaOchlp, C. albicans Ochl protein. Figure 5 is an illustration showing the gene recombination and pop-out to

induce the H. polymorpha OCHl gene disruption

Figure 6 shows the phenotype of the ochl defective mutant (Δochl) of H.

polymorpha. Yeast cells in a log phase were serially diluted 1 to 10, 5 μl was

spotted onto the YPD plate and the cells were cultured for 2 days. A, YDP media

a 37 ^°C ; B, YDP media at 45 °C ; C, YPD media containing 40 μg/ml of

hygromycin B; D, YPD media containing 0.4% of sodium deoxycholate; E, YPD

media containing 7 mg/ml of calcofluor white.

Figure 7 is a Western blot demonstrating the changes in the

oligosaccharide formation of glucose oxidase expressed in the H polymorpha

mutant, DL42-15, and the ochl defective strain (Δochl).

Description of the preferred embodiment

The present invention consists of selecting the naturally occurring sodium

vanadate-resistant mutant strain, DL42-15, originated from H. polymorpha DL-

1 ; cloning the H. polymorpha OCHl gene and analyzing the DNA sequence;

disrupting the H. polymorpha OCHl gene; testing for the glycosylation of the

Aspergillus niger glucose oxidase protein expressed in the sodium orthovanadate- .

resistant strain, DL42-15, and the defective mutant strain, Δochl. The invention describes engineering of the defective mutant, which was

mutated in the outer chain biosynthesis of a methylotrophic yeast H. polymorpha

to prevent hyperglycosylation by subsequent attachment of mannose residues.

This mutant is an ideal host for expression of human recombinant proteins

because it produces glycoproteins with fewer outer chains that more closely

resemble the native proteins and therefore do not initiate an immune response.

The hyperglycosylation-inhibiting mutants originated from H. polymorpha DL-1

were either a natural mutant selected by sodium orthovanadate or the mutant

mutated in the OCHl gene o H polymorpha.

The DNA sequence (nucleotide no. 1) of H. polymorpha OCHl cloned in

this study was deposited in GenBank (accession no. AF490971) and in the Korean

Collection for Type Culture (KCTC) on May 29, 2002 (accession no. KCTC

10265BP). The sodium orthovanadate-resistant strain, DL42-15, and the OCHl

gene-mutated strain, Δochl, of H. polymorpha were also deposited in the KCTC

on the same day (accession no. KCTC 10263BP and KCTC 10264BP, respectively).

This invention provides the DNA and amino acid sequences shown in Figure 3.

This invention provides the OCHl gene mutant (Δochl), which inhibits hyperglycosylation of glycoproteins.

This invention provides this mutant yeast strain as an expression host to

express genes encoding heterologous glycoproteins. This invention provides the hyperglycosylation-inhibiting mutant yeast

strain, DL42-15, deposited in KCTC (accession no. KCTC 10263 BP).

This invention provides this DL42-15 strain as an expression host to

express genes encoding heterologous glycoproteins.

This invention provides suitable conditions for cell culture of these

mutants as well as methods for the production and isolation of the recombinant

proteins from the culture.

Methylotrophic yeasts such as H. polymorpha and P. pastoris have been

extensively used for production of therapeutic recombinant proteins in medical

and pharmaceutical industries.

The term "hyperglycosylation-inhibiting" used in this study refers to

reduction of the oligosaccharide chains attached to glycoproteins expressed in the

mutants of the methylotrophic yeasts in comparison of those of the wild-type

yeasts.

The term "glycoproteins" used in this study refers to proteins processing

glycosylation on more than one residue of asparagine, serine or threonine of

glycoproteins in Hi polymorpha.

Possible glycoproteins that can be produced using these invented mutants

include, but are not limited to, the Aspergillus niger glucose oxidase, the S.

cerevisiae invertase, the HIV envelop protein, the influenza A virus

hemagglutinin, the influenza neuraminidase, the bovine herpes type-1 virus

glycoprotein D, the human angiostatin, erythropoietin, cytokine, human B7-1, B7-

2, B-7 receptor CTLA-4, human tissue factors, human growth factors (e.g. blood platelet-derived growth factor), tissue plasminogen activator, plasminogen

activator inhibitor-1, eurokinase, human lysosomal enzymes (e.g. α- galactosidase), plasminogen, thrombin, factor XIII and immune globulin. Those

glycoproteins can be used for therapeutic medicine delivered by injection, oral or

non-oral administration or other methods used in particular areas.

Glycoproteins produced in the mutants can be isolated and purified using

general methods for protein isolation and purification. However, the specific

methods employed depend on the property of the proteins to be isolated. These

properties should be determined by the parties interested. In brief, cultured cells

are collected, the secreted proteins are precipitated, and the proteins are isolated

and purified according to a general method for protein isolation and purification

using immune absorption, fractionation or chromatography

The following examples explain the invention in detail, however, the

claims are not limited to them.

Experimental example 1>

Selection of the sodium orthovanadate-resistant mutant strain, DL42-15, of

H. polymorpha

Even a low concentration (5 mM) of sodium orthovanadate generally

inhibits the growth of yeast. Most S. cerevisiae vanadate-resistantmutant

strainsare mutants with mutations in genes involved in glycosylation processing in the Golgi (Kanik-Ennulat et al., Genetics 140; 933-943, 1995); Uccelletti et al.

Res Microbiol 150:5-12, 1999). One of the most efficient methods for selection

for oligosaccharide biosynthesis defective mutants is using sodium orthovanadate

to select one with its resistance and this method has been extensively used in S.

cerevisiae and Kluyveromyces lactis. However, this method cannot be used in the

methylotrophic yeast P. pastoris because it itself is resistant to sodium

orthovanadate (Martinet et al., Biotechnology Lett. 20, 1171-1177, 1999). In the

case of another methylotrophic yeast H. polymorpha, CBS 4732 and NCYC 495

strains have also been reported that they can grow in the media containing 96 mM

sodium orthovanadate (Mannazzu et al., FEMS Microbiol Lett. 147: 23-28, 1997;

Mannazzu et al. Microbiology 144: 2589-2597, 1998).

The H. polymorpha DLl, used in this study to develop a expression host

for production of recombinant proteins, showed a similar sensitivity to sodium

orthovanadate to S. cerevisiae unlike CBS 4732 and NCYC 495 (Figure 1 and

Table 1). The natural mutant cells of H. polymorpha DLl, which became

resistant to the sodium orthovanadate , occurred at a frequency of 1 perlO⁶ cells

on the YPD media plate containing 4 mM sodium orthovanadate. showed that

This mutation frequency is similar to that in the wild type S. cerevisiae (Table 1).

[Table 1]

Growth comparison of yeast strains grown on the YPD plates containing sodium orthovanadate.

* The results were obtained after culturing at 30^°C (S. cerevisiae)ox at 37°C (H.

polymorpha) for 4 days.

All the defective mutants of oligosaccharide biosynthesis among the

sodium orthovanadate-resistant mutants of S. cerevisiae have been shown to be

more sensitive to antibiotics with a large molecular- weight such as

aminoglycoside, to synthetic detergents such as sodium deoxycholate, and to high

temperature (Dean N., Proc. Nαtl. Acαd. Sci. USA 92, 1287-1291, 1995). We

selected 250 natural mutants from H. polymorpha DLl showing more resistance

to sodium orthovanadate, most (over 90%>) of which were also resistant to

hygromycin B. The selected mutants have been further tested on the media

containing sodium orthovanadate at high temperature (45 °G>) to select the mutant

colonies resistant to sodium orthovanadate but sensitive to high temperature. Finally, the mutants have been isolated and designated as H polymorpha DL42-

15 (Figure 2 and 3).

Experimental example 2>

Cloning and DNA sequence analysis of the H. polymorpha OCHl gene We analyzed the Random Sequenced Tags (RSTs) of the partial genomic

analysis of H. polymorpha (Blandin et al, FEBS Lett. 487, 76, 2000) and

obtained the partial DNA sequences of genes showing homology with the genes

involved in the oligosaccharide biosynthesis of S. cerevisiae. The predicted

amino acid sequences deduced from the partial DNA sequences share homology

with a region corresponding to the C-termini of S. cerevisiae OCHl (ScOCHl),

which plays an important role in attachment of αl, 6-mannose in the beginning of

the outer chain biosynthesis. S. cerevisiae ScOCHl also shares high homology

to S. cerevisiae HOCl (ScHOCl). A pair of primers designed based on the partial

DNA sequences are 5'-CAATCAGACCCGGTCTGTCGAGGAGT-3'(nucleotide

no. 3), 5*-ACATCAACGTGGAGAACTGGGAGCAC-3' (nucleotide no. 4). Using

these primers, we amplified by PCR a 900 bp fragment from genomic DNA

isolated from H. polymorpha.

We performed Southern blotting, probed with the 900 bp fragment, using the genomic DNAs digested with several restriction enzymes. In order to isolate

the promoter region and full-length of the H. polymorpha OCHl gene, we gel-

extracted the two fragments of 2.3 kb (digested with BamHI) and 5 kb (digested

with Bglll) corresponding to the signals of the Southern blot. Each fragment was

then cloned into a cloning vector pBluescript KS+ (Stratagen Co.). The clones

were sequenced in both strands.

The DNA sequence analysis revealed the clones include the promoter

region of 1 kb and the open reading frame of 1.3 kb encoding a putative protein with 435 amino acids (nucleotide no. 1, Figure 3). The predicted protein of H.

polymorpha was designated as HpOchl (amino acid sequence no.2). This protein

shares low homology (21-23%) to ScOCHl (accession no. YGL038C), ScHOCl

(accession no. YJR075W) and Candida albicans Ochl (accession no. AY064420)

proteins. However, it contains a DXD motif, a possible activation site, and the

transmembrane spanning region in the N-terminal found in the

mannosyltransferase, a type II membrane protein (Figure 4).

Experimental example 3>

Production and analysis of the OCHl gene-mutated strain ( Δ ochl) of H

polymorpha

In order to make the mutants where the OCHl gene was disrupted, two

techniques, fusion PCR using the primers listed in Table 2 and in vivo DNA

recombination, were used for the gene disruption (Oldenburg et al, Nucleic Acid

Res. 25, 451, 1997). The regions corresponding to the N-terminal and the C-

terminal of URA3 and OCHl genes, respectively, were amplified by PCR. The

fragment corresponding to the N-terminal of HpOCHl was then fused by fusion

PCR to the fragment corresponding to the N-terminal of URA3 while the fragment

corresponding to the C-terminal of HpOCHl was fused to the fragment

corresponding to the C-terminal of URA3. The fused DNA fragments were

introduced into yeast cells to make recombination of the gene. Transformants where the HpOCHl gene was disrupted were then selected (Figure 5). The

mutants were first screened on the minimal media containing no uracil, selecting

for the URA3. marker. PCR was then performed on the genomic DNAs isolated

from the mutants and the wild type to confirm the HpOCHl gene disruption. An

H. polymorpha mutant Δochl(leu2 ochl::URA3) was selected based on analysis

of the PCR products.

The selected mutant strain Δochl grows more slowly than the wild type; it

is more sensitive to a high temperature of 45 ^°C and to hygromysin B; its growth

is inhibited by addition of sodium orthovanadate and calcofluor white (Figure 6).

All these properties are common in the defective mutant strains of the outer chain

biosynthesis in yeasts, suggesting the mutant strain Δochl has a defect in the

biosynthesis.

[Table 2]

' Primers used in this study for PCR to disrupt the HpDCHl gene

Experimental example 4>

Analysis of the recombinant glycoproteins expressed and isolated from the mutant

strains, DL42-15 and Δochl, of H. polymorpha.

In order to examine the glycosylation defect on a recombinant

glycoprotein expressed in the mutant strains, DL42-15 and Δochl, described in

experimental example 1 and 3 respectively, we expressed the glucose oxidase

(GOD) of an Aspergilhis niger gycoprotein in these mutants. The GOD protein

contains the 8 potential sites for the N-linked glycosylation (Frederick et al, J.

Biol Chem. 265, 3793, 1990).

In order to express the GOD in the mutant yeast strains, we constructed a

GOD expression vector, pDLMOX-GOD using the pDLMOX-Ηir vector (Kang et

al Yeast 14, 371, 1998)). The DNA fragment containing the hirudin gene was

first removed from the pDLMOX-Ηir vector and the GOD gene fused to the

fragment corresponding to the secretory signal of the α-amylase at the N-terminal

was then replaced in the vector (Kim S. Y. Ph. D. Dissertations, Yonsei University, Korea, 2001). The resultant vector pDLMOX-GOD was introduced

into the two mutant strains, DL42-15 and Δochl as well as the wild type strain, and they were cultured on the YPM media (1% yeast extract, 2% peptone, 2%

methanol) to express the GOD proteins.

The GOD proteins expressed and secreted were isolated and purified for

Western blot anlaysis. The proteins were run on a polyarcylamide gel, transferred

to a nitrocellulose membrane, and blotted using a GOD antibody. Figure 7A

shows that the GOD proteins of the mutant strains, DL42-15 and Δochl, have a

smaller molecular weight than that of the wild type, suggesting the proteins

expressed and secreted in the mutants are less hyperglycosylated, or in other

words, hyperglycosylation is inhibited in the mutant strains. To confirm the

blotting result, we treated all the proteins with endoglycosidase H enzyme to

digest the oligosaccharide chains attached on the proteins, and repeated the blot.

Figure 7B shows that all the proteins have the same molecular weight on the blot,

suggesting they are all the same proteins. These results demonstrate that the

proteins expressed and secreted in the mutant cells were smaller than the one

expressed and secreted in wild type cells due to less hyperglycosylation on the

proteins. Therefore, the mutant strains, DL42-15 and Δochl, unlike the wild type,

are suitable host cells to produce the human glycoproteins, in which the

hypergylcosylation of the proteins will be inhibited, resulting in a closer resemblance to native human proteins.

Possible application of the invention to industries The H. polymorpha mutants, DL42-15 and Δochl, axe able to be used as

host cells to produce recombinant glycoproteins, which will express and secrete

the proteins containing proper outer oligosaccharide chains closely resembling

the native proteins because the hyperglycosylation of the proteins is inhibited in

the mutants cells. These mutants will be useful in the medical therapeutic industry

because H. polymorpha yeast cells has been broadly used to produce medical

therapeutic recombinant proteins on a large scale.

INDICATIONS RELATING TO DEPOSITED MICROORGANISM

OR OTHER BIOLOGICAL MATERIAL

(PCT Rule 13 bis)

A. The indications made below relate to the deposited microorganism or other biological material referred to in the description on page 8 . line 12-13

B. IDENTIFICATION OF DEPOSIT Further deposits are on an additional sheetD

Name of depositary institution Korean Collection for Type Cultures

Address of depositary institution(inchiding postal code and country)

#52, Oun-dong, Yusong-ku, Taejon 305-806, Republic of Korea

Date of deposit Accession Number 29/05/2002 KCTC I0265BP

C. ADDITIONAL INDICATIONS (lecnebhnkffhotcφplicώk) This information is continued on an additional sheet D

D.DESIGNATED STATES FOR WHICH INDICATIONS ARE UADΕ.(if the indications are not for all designated States)

E.SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable)

The indications listed below will be submitted to the International Bureau l&ter(specijy the general nature of the indications e.g., "Accession Number of Deposit")

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File reference PCTA/KRIB/2 PCT/KR03/01285

INDICATIONS RELATING TO DEPOSITED MICROORGANISM

OR OTHER BIOLOGICAL MATERIAL

(PCT Rule 13bιs)

For receiving Office use only For international Bureau use only

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INDICATIONS RELATING TO DEPOSITED MICROORGANISM

OR OTHER BIOLOGICAL MATERIAL

(PCT Rule 13bis)

A. The indications made below relate to the deposited microorganism or other biological material referred to in the description on page 8 . line 14-15

B. IDENTIFICATION OF DEPOSIT Further deposits are on an additional sheetD

Name of depositary institution Korean Collection for Type Cultures

Address of depositary institution(inc luding postal code and country)

#52, Oun-dong, Yusong-ku, Taejon 305-806, Republic of Korea

Date of deposit Accession Number 29/05/2002 KCTC 10264BP

C. ADDITIONAL INDICATIONS (leave blank fnotqpl able) This information is continued on an additional sheet O

D.DESIGNATED STATES FOR WHICH INDICATIONS ARE UADE(ifthe indications are notfor all esignated States)

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The indications listed below will be submitted to the International Bureau later(specijy the general nature of the indications e.g., "Accession Number of Deposit")

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Claims

What Is Claimed Is:

1. A nucleic acid molecule comprising the DNA sequence shown in Figure 3.

2. The nucleic acid molecule according to claim 1, wherein the nucleic acid

molecule is Hansenula polymorpha HpOCHl gene (KCTC 10265BP).

3. A polypeptide comprising the amino acid sequence shown in Figure 3.

4. H. polymorpha mutant strains which prevent the hyperglycosylation of

glycoproteins by mutation of the HpOCHl gene.

5. A H. polymorpha mutant strain Δochl (KCTC 10264BP) according to claim 4,

wherein the HpOCHl gene is disrupted. .

6. A recombinant H. polymorpha strain expressing a foreign protein, wherein the

recombinant strain is produced by introducing the gene encoding the foreign

protein into H. polymorpha strain according to claim 4.

7. A recombinant H. polymorpha strain according to claim 6 expressing a foreign

protein, wherein the recombinant strain is produced by introducing the gene

encoding the foreign protein into the disrupted mutant strain, Δochl .

8. A natural mutant strain, DL-42-15, originated from H. polymorpha DLl strain,

prevented from hyperglycosylation of glycoproteins (KCTC 10263BP).

9. A recombinant H. polymorpha strain expressing a foreign protein, wherein the

recombinant strain is produced by introducing the gene encoding the foreign

protein into H. polymorpha mutant strain, DL-42-15 according to claim 8

10. A method for producing a foreign protein, wherein the method comprises

cultivating the recombinant strains according to any one of claims 6, 7, and 9

under conditions that allow the strains to express the foreign protein and isolating

the expressed foreign protein from the cultures.

11. The method according to claim 10, wherein hyperglycosylation of a foreign protein is prevented.