KR0161148B1 - Preparation method for heterologous protein by recombinant methylotrophic yeast - Google Patents

Abstract

The present invention relates to a method for producing a heterologous protein from methanol magnetized yeast (methylotrophic yeast), according to the method of culturing by adding oil to a recombinant methanolized magnetized yeast culture medium containing a heterologous protein gene to improve the productivity of the heterologous protein In particular, using a methanol oxidase (MOX) promoter and an expression vector comprising an MOX terminator sequence and a hirudin gene, methanol magnetization yeast can be efficiently produced.

Description

Production Method of Heterologous Protein Using Recombinant Methanol Magnetized Yeast

Figure 1 shows the manufacturing process of the expression vector pC782 comprising the hirudin gene and Hansenula auto-replicating sequence HARS3 of the present invention,

Figure 2 shows the manufacturing process of the expression vector pA2-HG comprising the hirudin gene and Hansenula auto-replicating sequence HARS2.

The present invention relates to a method for producing a heterologous protein from methanol magnetotrophic yeast, and more particularly, to a method for improving the productivity of the heterologous protein by adding oil to a recombinant methanol magnetizing yeast culture medium, in particular methanol oxidase. (Methanol Oxidase: MOX) The present invention relates to an expression vector comprising a promoter, a MOX terminator sequence and a hirudin gene, a methanol magnetized yeast transformed with the expression vector, and a method for efficiently producing hirudin by culturing the transformant. .

Hansenula polymorpha and Pichia pastoris, the most representative examples of methanol magnetizing yeast, are alcohol oxidase (AOX) and methanol oxidase (MOX) gene promoters, respectively. The development of heterologous protein expression systems using a methanol-induced promoter called `` Buckholz, RGand MAGGleeson, Yeast systems for the commercial production of heterologous proteins. Bio / Technology, 9, 1067-1072 (1991)). The foreign protein expression system using methanol magnetization yeast has the advantage that it is more economical than Saccharomyces cerevisiae, which uses methanol as a carbon and energy source to express foreign proteins using sugar.

This study on methanol magnetizing yeast is mainly conducted on the study of methanol metabolism (Dijken JP van, W. Harder, AJ Beardsmore and JR Quayle, Dihydroxyacetone: an intermediate in the assimilation of methanol by yeast? FEMS Microbiol. Lett., 4, 97-102 (1978)), and studies of related enzymes (Egli, T., JP van Dijken, M. Veenhuis, W. Harder and A. Fichter, Methanol metabolism in yeast: regulation of the synthesis of catabolic enzymes, Arch Microbiol., 124, 115-121 (1980)) and studies on growth characteristics (Dijken, JP van, R. Otto and W. Harder, Growth of Hansenula polymorpha in a methanol-limited chemostat.Physiological responses due to the involvement Many studies have been conducted, such as of methanol oxidase as a key enzyme in methanol metabolism, Arch. Microbiol., 111, 137-144 (1976), and since 1980, optimization of cell culture (Egli, TH and JR Quayle) , Influence of the carbon: nitrogen ratio of the growth medium on t he cellular composition and the ability of the methylotrophic yeast Hansenula polymorpha to utilize mixed carbon sources, J. Gen. Microbiol., 132, 1779-1788 (1986)), metabolite production studies (Kato, N., H. Kobayashi, M) Shimao and C. Sakazawa, Dihydroxyacetone production from methanol by a dihydroxyacetone Kinase deficient mutant of Hansenula polymorpha, Apppl. Microbiol. Biotechnol., 23, 180-186 (1986)) and high concentration culture studies for single cell protein (SCP) production (Wegner, EH, Biochemical conversions by yeast fermentation at high cell densities, US Pat. No. 4,414,329 (1983)). Application research using the function of the strain itself has begun.

A study to use methanol magnetized yeast as a host cell for recombinant protein production has been studied for hepatitis B surface antigen vaccine production (Cregg, JM, JF Tschopp, C. Stillman, R. Seigel, M. Akong). , WS Craig, RG Buckholz, KR Madden, PA Kellaris, GR Davis, BL Smiley, J. Cruze, R. Torregrossa, G. Velicelebi and GP Thill, High-level expression and efficient assembly of hepatitis B surface antigen in the methylotrophic yeast , Pichia pastoris, Bio / Technology, 5, 479-485 (1987)), and in the late 1980s the production of epidermal growth factor (FGF) (Siegel, RS, R. Buckholz and GP Thill, Production of epidermal growth factor in Pichia pastoris yeast cells, European patent application WO 90/10697), production of glucoamylase (Gellissen, G., ZA Janowicz, a. Merckelbach, M. Piontek, P. Keup, U. Weydemann, CP Hollenberg and AWM Strasser, Heterologous gene expression in Hans enula polymorpha: Efficient secretion of glucoamylase, Bio / Technology, 9, 291-295 (1991), production of streptokinase (Hagenson, MJ, KA Holden, KA Parker, PJ Wood, JA Cruze, M. Fuke, TR Hopkins and DW Stroman, Expression of streptokinase in Pichia pastoris yeast, Enzyme Microb. Technol., 11, 650-656 (1989)) and production of alpha-galactosidase (Fellinger, AJ, JMA Verbakel, RA Veale, PE Sudbery, IJ Bom, N. Overbeeke and CT Verrips, Expression of the α-galactosidase from Cyamopsis tetragnoloba (guar) by Hansenula polymorpha, Yeast, 7, 463-471 (1991)) has produced a number of recombinant proteins.

As mentioned above, many studies on the expression system of foreign genes using methanol magnetizing yeast have been conducted. On the other hand, studies on the development of new fermentation techniques for efficient production of heterologous proteins have been rarely reported. In general, the method of culturing the cells as high concentration as possible using glucose or glycerol (carbonol) as a carbon source and then supplying methanol to induce gene expression (Digan MESV Lair, RA Brierley, RS Siegel, ME) Williams, SB Ellis, PA Kellaris, SA Provow, WS Craig, G. Velicelebi, MM Harpold, Continuous production of a novel lysozymevia secretion from the yeast, Pichia pastoris, Bio / Technology, 7, 160-164 (1989)) have. The more modified production methods here include three stage fermentation methods using mixed carbon sources (Brierley, RA, RS Siegel, CM Bussineau, WS Craig, GC Holtz, GR Davis and RG Buckholz, Mixed feed recombinant yeast fermentation, PCT WO 90 / 03431 (1990)), in the first step, using glycerol as the only carbon source, incubated until a certain cell concentration was obtained, and in the second step, limited supply of glycerol to maintain a very low concentration of methanol metabolism After inducing sufficient release, the third step is a method of inducing high expression of a foreign gene by continuously supplying a glycerol-methanol mixed substrate which gradually increases the ratio of methanol.

Hansenula polymorpha, a methanol magnetizing yeast, uses HARS (Autonomously Replicating Sequences of Hansenula), a gene essential for the stable maintenance and expression of foreign protein genes introduced into host cells. ) Is possible. HARS is present in the host cell chromosome at about 30kb intervals, and they are known to have different levels of ARS activity. Therefore, in order to develop a recombinant protein production system using Hanshenula polymorpha, ARS is selected from among a large number of HARSs present in the chromosome, and at the same time, ARS having excellent function of multiple replication integration of heterologous genes is selected. We need to develop a vector that can introduce heterologous genes into the host and maintain them in multiplex. In addition, this vector should be used to develop a recombinant Hansenula polymorpha system suitable for foreign protein production, and finally to develop a fermentation process for recombinant protein using the strain.

Since the recombinant protein production system using Hansenula polymorpha is a system for expressing heterologous genes using MOX promoter, it is necessary to supply a large amount of methanol to fermentation medium for a long time during fermentation. However, while methanol is an essential ingredient as an expression inducer, it has a double side effect that negatively affects cell activity in the presence of excess, and by-products such as formic acid and formaldehyde generated during methanol metabolism also negatively affect cell activity. Prolonged fermentation leads to cell deactivation. In addition, there is a problem that productivity is lowered due to degeneration of the protein produced during methanol fermentation.

Another problem with using recombinant methanolized yeasts as host cells is the overuse of methanol due to potent MOX activity. In general, the optimum growth temperature of methanol magnetizing yeast is 38 ℃. At this temperature, the activity of MOX is strong. In addition to using methanol as a carbon and energy source, it is also used as an enzyme reaction substrate for MOX. Therefore, the heterologous protein expression system using recombinant methanol magnetizing yeast, which maintains methanol concentration above a certain level, induces MOX promoter, requires more methanol and generates excess formaldehyde and hydrogen peroxide in proportion. Toxic hydrogen peroxide continues to be broken down into water and oxygen by catalase, which increases the volume of fermentation broth due to the production of large amounts of water, up to about 70% of the volume of methanol supplied. This dilution ultimately leads to a decrease in productivity. Therefore, the development of a fermentation process that can reduce the amount of methanol possible and increase the expression rate is required.

Accordingly, the present inventors have continued to develop a fermentation technology that stably and efficiently produces heterologous proteins from methanol magnetized yeast. As a result, the inventors have found that the addition of oil to fermentation medium can increase the stability and productivity of fermentation. In particular, by culturing the Hansenula polymorpha strain transformed with an expression vector including the MOX promoter and the MOX terminator sequence and the hirudin gene, it was found that hirudin can be produced with high efficiency.

Accordingly, it is an object of the present invention to provide a method for efficiently producing heterologous proteins by increasing the stability and productivity of fermentation when expressing heterologous genes using methanol magnetized yeast as host cells.

Another object of the present invention is to provide an expression vector capable of producing hirudin with high efficiency from methanol magnetizing yeast and a methanol magnetizing yeast transformed with the expression vector.

In order to achieve the above object, the present invention provides a method for producing a heterologous protein by orienting a recombinant methanol magnetizing yeast containing a heterologous protein gene, the method for producing a heterologous protein, characterized in that the culture by adding oil to the medium to provide.

In order to achieve the above another object, in the present invention, an expression vector comprising a methanol oxidase promoter, a preproderer, a hirudin gene, a methanol oxidase terminator sequence and a Hanshenula autoreplication sequence HARS3, and a transformed vector with the expression vector Provides methanol magnetization yeast.

Hereinafter, the present invention will be described in more detail.

Expression vectors of the present invention for efficiently producing heterologous proteins, such as hirudin, from Hanshenula polymorpha, a type of methanol magnetizing yeast used in the present invention, include MOX promoter, preproder, hirudin gene, and MOX terminator. And HARS3, for example pC782 can be manufactured as follows. First, a plasmid pCKS-Cmox3, a hirudin expression cassette containing a cross-linking factor α-secretion signal and a hirudin gene of the MOX promoter and Saccharomyces cerevisiae, was treated with restriction enzymes to obtain plasmid pCmox-ΔXhoI. The plasmid pMHC7 is obtained by linkage after restriction enzyme treatment with plasmid pYELC5 containing CUP1, a copper resistance gene derived from Seth cerevisiae. Subsequently, the plasmid p82A3 containing the self-replicating sequence HARS3 of the Hanshenula polymorpha strain and the pMHC7 were treated with a restriction enzyme and linked to obtain plasmid pC782.

Plasmid pC782 contains the MOX promoter-preproderer-hirudin gene-MOX terminator in this order, including the HARS3 of Hansenula polymorpha strain and the copper resistance gene (CUP1) of Saccharomyces cerevisiae. Multiple replication selection is easy with a degree of resistance to copper.

Hirudine can be efficiently produced by transforming a methanol magnetized yeast, such as Hanshenula polymorpha DL-1 (leu2), with the recombinant expression vector pC782 prepared as described above and culturing under suitable conditions on a suitable medium.

The heterologous protein production method of the present invention is characterized by culturing by adding oil (oil) to the culture medium when culturing the recombinant methanol magnetizing yeast containing the heterologous protein gene. Oil not only increases the expression level of heterologous proteins, but also appears to prevent protein denaturation. Oils that can be used include vegetable cooking oils such as soybean oil, corn germ oil, rapeseed oil and cottonseed oil, which are preferably added at a concentration of 0.5 to 2% in the medium.

In addition, in the heterologous protein production method of the present invention, an antioxidant may be added to the medium to prevent rancidity of the oil. At this time, tocopherol, astaxanthin, and the like can be used as the sulfating agent, and it is preferable to add it in a concentration of 0.01 to 0.1% in the medium.

Hereinafter, the present invention will be described in more detail by the following examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.

Example 1

Preparation of hirudin expression vector pC782 containing HARS3 and hirudin expression vector pA2-HG comprising HARS2

Plasmid pBKS-Cmox3 (Sohn, JH, MY Beburov, ES Choi and SK Rhee, Heterologouse gene expression, a hirudin expression cassette containing a cross-linking factor α-secretion signal and hirudin gene of MOX promoter and Saccharomyces cerevisiae and secretion of the anticoagulant hirudin in a methylotrophic yeast Hansenula polymorpha, J. Microbiol. Biotechnol., 3, 65-72 (1993)) were digested with restriction enzyme XhoI to obtain plasmid pCmox-ΔXhoI. Plasmid pYELC5 (Macreadie, IG, Nucl. Acids Res., 18, 1078 (1989)) containing CUP1, a copper resistance gene derived from Saccharomyces cerevisiae, was cleaved with the restriction enzyme HindIII and cleno After treatment, the residue was cleaved with SacI, and the plasmid pCmox-ΔXhoI obtained above was cleaved with EcoRV and SacI and ligated to obtain plasmid pMHC7. This plasmid pMHC7 was treated with restriction enzymes ScaI and SacI, and plasmid p82A3 (Bogdanova, AI, MO Agaphonov and MD Ter-Avanesyan, Plasmid reorganization during integrative transformation in Hansenula polymorpha) containing the self-replicating sequence HARS3 of Hanshenula polymorpha strain. , Yeast, 11, 343-353 (1995)) were treated with restriction enzymes ScaI and SacI and linked to obtain plasmid pC782 (see FIG. 1).

The procedure was carried out according to the method of Maniatis et al. (Maniatis T., EF Fritsch and J. Sambrook, Molecular cloning. A laboratory manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New york, USA). . Restriction enzymes, ligase and the like were purchased from New England Biolab.

On the other hand, plasmid pA2-HG containing HARS2 instead of HARS3 of Hanshenula polymorpha strain was prepared by the following method.

Plasmid pMHC7 (see Figure 1) was digested with restriction enzymes XhoI and ScaI, clenotreated, and then DNA fragments containing hirudin genes were isolated and plasmid pA2 containing HARS2 (Bogdanova, AI, MO Agaphonov and MD Ter). -Avanesyan, Plasmid reorganization during integrative transformation in Hansenula polymorpha, Yeast, 11, 343-353 (1995)) was treated with restriction enzymes HindIII and ScaI and treated with Klenow to connect with DNA fragments containing the hirudin gene to plasmid pA2- HG was obtained (see Figure 2).

Example 2

Transformation of Hanshenula polymorpha DL-1 (leu2)

Sohn, JH, MY Beburov, E, S. Choi and SK Rhee, Heterologouse gene expression and secretion of the anticoagulant hirudin in a methylotrophic yeast Hansenula polymorpha, J. Microbiol. Biotechnol., 3, 65-72 ( 1993)), plasmids pC782 and pA2-HG prepared in Example 1 were used to transform Hansenula polymorpha DL-1 (leu2) from MY Beburov, NPO biotechnologia, Russia, respectively.

The transformants were incubated for 48 hours at 37 ° C. in YND medium (yeast nitrogen base without amino acid 0.67%, dextrose 2%) containing 0.6 mM of copper, and the growth rate was high and high hirudin expression Hansenula poly Morfa DL1-008 was selected and deposited on July 15, 1995, as a deposit number KCTC 8677P to the Genetic Bank (KCTC), an affiliated biotechnology research institute of Korea Institute of Science and Technology.

The method used for hirudin analysis in the examples is as follows:

Dilute human thrombin (Sigma) to 0.6NIH U / ml in thrombin reaction buffer consisting of 0.1M Tris-HCl (pH 8.0), 0.12M NaCl, 0.01% sodium azide and 0.1% bovine serum albumin, Chromozym TH (Boehringer Mannheim), an artificial substrate of thrombin, was also dissolved in thrombin reaction buffer to 200 μM. Standard hirudin (Accurate Chemical and Scientific Corporation) was used diluted in concentrations of 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6 ATU (Anti-thrombin unit) / ml in thrombin reaction buffer. Where 1 ATU represents the amount of hirudin that completely inhibits human thrombin 1 NIH units. 50 μl of thrombin solution is added to each well of a 96-well microtiter plate of 50 μl each of standard hirudin and appropriately diluted unknown sample, and 100 μl of chromozyme TH solution is added to The amount of hirudin was determined by measuring the increase in absorbance at 405 nm for 5 minutes using ELISA reader: THERMO max, Molecular Devices).

Example 3

Hirudin expression level and HARS activity

Plasmid pC782 comprising HARS3 obtained in Example 2 and plasmid YEp13 having LEU2 gene derived from Saccharomyces cerevisiae and Hansenula polymorpha strains transformed with pA2-HG comprising HARS2 Table 1 shows the comparison of hirudin expression rates of the isolated strains. Next, the fast-growing transformants were selected, and the integration form, the copy number, and the hirudin activity were examined by Southern hybridization, and their correlations are shown in Table 2 below. The probe used for DNA hybridization was a DNA labeling and detection kit from Boehringer Mannheim, and the hybridization was performed using a hybridization oven at 42 ° C. in a hybridization solution containing 50% formamide. All strain cultures were performed in a 38 ° C. shake incubator using a 500 ml Erlenmeyer plasmid. In the spawn culture, YNG medium (yeast nitrogen base without amino acid 0.67%, glycerol 2%, pH 5.5), YPM medium (1% yeast extract, 2% peptone, 1% methanol, pH 5.5) were 121 ° C, respectively. It was used after sterilization for 15 minutes. Fermentation was performed by adding 1 ml of the spawn culture medium to 50 ml of YPM medium and shaking culture at 38 ° C. for 24 hours, supplementing with 1% methanol, incubating for 24 hours, and then measuring the amount of hirudin expression.

In Table 1 transformants 1 to 35 were obtained using plasmid pC782 cut with restriction enzymes HindIII and XhoI, 36 and 37 were obtained using cyclic plasmid pC782, and 38 had both hirudin and CUP1 as controls. It was obtained using the cyclic plasmid pA2 not present. As shown in Table 1, the stronger the resistance to copper, the stronger the hirudin activity was.

On the other hand, transformants using plasmid pC782 were randomly integrated with hirudin expression cassettes of several copies (4 to 5 copies), and transformants using pA2-HG had a small number of copies but tandems. tandem). As shown in Table 2, the amount of hirudin expression in the transformants containing HARS3 was the best.

Example 4

Effect of oil addition on hirudin expression

Among the transformants tested in Example 3, a transformant having the highest hirudin expression rate was selected and named Hansenula polymorpha DL1-008 and hirudin expression was performed using this strain.

To investigate the effect of oil addition on hirudin expression, in YGM medium (2% yeast extract, 5% glycerol, 1% methanol) and YGMF medium (YGM medium + oil 1%), respectively, cooking oil (soybean oil) Incubated under the same conditions as in Example 3. However, by adopting a fed-batch culture method, 1% (0.5ml) of methanol was added every 12 hours from 48 hours after the start of the culture to induce continuous expression of hirudin. As shown in Table 3, the concentration of hirudin reached up to 135.8 mg / L with continuous expression when oil was added, but only up to 55.4 mg / L when no oil was added. It turns out to have a significant effect.

Example 5

When methanol is added during hirudin expression

In the YGMF medium and YMG medium used in Example 4, respectively, except for methanol, YGF medium and YG medium, hirudin expression was carried out by culturing differently in the same conditions as in Example 4 only methanol addition time. As the strain, the same Hansenula polymorpha DL1-008 as in Example 4 was used. Methanol was first added 1% (0.5ml) 36 hours after the start of expression, and then 1% (0.5ml) was added every 12 hours.

As shown in Table 4, the initial addition time of methanol did not affect the production of hirudin. However, as in Example 4, up to 138.4 mg / L of hirudin was produced in the oil-added YGF medium, while up to 32.3 mg / L of hirudin was produced in the oil-free YG medium.

Example 6

Hirudin expression with oil or antifoam

In order to test whether the oil addition effect in hirazine expression is simply due to the role of the antifoaming agent of oil, hirudin expression is performed using a medium containing neoin, one of the commonly used antifoaming agents. The results were compared.

Cultures were added to YGM medium (1% yeast extract, 2% glycerol, 1% methanol) by adding 1% oil or 0.1% neoline, respectively, and strains were used as Hansenula polymorpha DL1-008. Methanol was added in 1% increments every 12 hours from the beginning of 40 hours. The concentration of hirudin produced after 90 hours of expression was 13.0 mg / L with YGM medium without oil or neoin and 27.2 mg / L with oil. In the case of 7.0mg / L, the antifoaming agent was found to have a negative effect on the expression of hirudin.

Example 7

Role of oil in hirudin expression

In order to investigate the role of oil in the expression of hirudin, the hirudin sample obtained by the expression time zone in Example 5 was partially purified by Sep-Pak (Mip-Pak, Millipore) to fix the C-8 column (4.6 × 250 mm). Analysis using high pressure liquid chromatography (HPLC).

In Table 5, the standard hirudin peak was found at the residence time of 24.5 minutes, and even in the case of the sample using the YGF medium, the main peak appeared at the same position as the standard sample. On the other hand, in the case of the sample using the oil-free YG medium, the main peak was minute, while the peak estimated to be denatured hirudin was relatively large around the hirudin main peak.

In addition, the activity of the main hirudin in the antithrombin (antithrombin) activity was about 300 times higher than the activity of the denatured hirudin. On the other hand, in the case of YGF, the modified hirudin peak began to appear in the sample after 144 hours after the late expression hirudin concentration began to decrease. From these results, it was confirmed that in addition to increasing the expression rate of hirudin by the addition of oil, degeneration of hirudin was also prevented for a considerable period of time.

Example 8

Expression of hirudin with antioxidant

In the case of oil-induced hirudin expression, there is a case in which the rancidity of the oil is halved due to the long-term aeration culture. Culture was added by adding 0.1% in the medium. Tocopherol was used after dissolving in methanol at 1% concentration for 24 hours to facilitate sterilization and addition. Table 6 shows the results of hirudin expression in Hanshenula polymorpha DL1-008 and YGMFT medium (YGMF medium + tocopherol 0.1%) under the same conditions as in Example 4.

As shown in Table 6, the resulting primary hirudin concentration was 104.5 mg / L, which was lower than that of the oil-only YGMF medium, but no oil rancid occurred when the YGMF medium was used. A decrease in concentration was also prevented.

Example 9

Hirudin expression using media with increased oil concentration

Table 7 shows the results of hirudin expression incubated using YGMFT medium having an oil concentration increased to 2% in Example 4. Increasing the oil concentration to 2% did not inhibit cell growth or hirudin production, and the maximum hirudin production concentration was 107.6 mg / L, which was slightly higher than that of 1% oil.

Example 10

Hirudin expression using medium with increased antioxidants

In Example 8, the results of hirudin expression cultured using a medium in which the tocopherol concentration was increased to 0.2% are shown in Table 8. In this case, the maximum hirudin concentration was 80.3 mg / L, and the use of excess tocopherol inhibited cell growth and hirudin production.

Example 11

Hirudin expression using media with increased concentrations of oils and antioxidants

In Example 8, the results of hirudin expression cultured using a medium in which the tocopherol concentration was increased to 0.2% and the oil concentration to 2% are shown in Table 9. In this case, the maximum hirudin concentration was 100.4 mg / L, which was lower than that of using 1% oil and 0.1% tocopherol, but did not show the same inhibition as when only the concentration of tocopherol was increased.

Example 12

Expression of hirudin at 30 ° C.

Table 10 shows the results of performing hirudin expression under the same conditions as in Example 4 except that the cultivation was carried out using YGMFT medium at 30 ° C.

The maximum hirudin concentration was 93.1 mg / L, which was slightly lower than that at 38 ° C., but the denaturation of hirudin did not occur much. Therefore, in order to reduce excessive consumption of methanol by reducing MOX activity during fermentation with hirudin, Expression at ° C was judged to be effective.

Example 13

Hirudin expression using fermenter

5 ml containing 150 ml of Hansenula polymorpha DL1-008 shaken at 30 ° C. for 24 hours using YNG media, and 2850 ml of YPGMFT medium (YGMFT medium + 2% peptone) fortified with 10% glycerol concentration. It was inoculated into a Bio Flow III (NBS) fermenter. The temperature of the fermenter was adjusted to 30 ℃ and pH was adjusted to 5.5 using 50% ammonia water and 4N-HC1. For aerobic aeration culture, while stirring the medium at 500 to 1000rpm, 3L of sterilized air was supplied per minute so that the dissolved oxygen concentration in the medium did not fall below 2%. After glycerol in the medium was consumed completely, methanol was fed continuously at 10-20% per hour. All fermentation conditions as described above were carried out by a computerized control method using a computer, it is shown in Table 11 by measuring the hirudin production in each time zone.

As shown in Table 11, 160 hours of expression was performed while maintaining the methanol concentration in the medium at 1 to 4%. As a result, 318 mg of hirudin per liter medium was obtained. This is an increase of almost 10 times compared to 30 to 40 mg / l obtained by the conventional method.

As described above, when methanol magnetizing yeast of the present invention is used as a host cell, heterologous proteins such as human epidermal growth factor and urokinase can be produced with high efficiency as well as hirudin.

Claims (8)

A recombinant methanol magnetized yeast transformed with an expression vector comprising a methanol oxidase promoter, a preproleader, a heterologous protein gene, a methanol oxidase terminator sequence and a Hanshenula autoreplication sequence HARS3 is cultured in a medium containing plant oil. Method of producing heterologous protein. The method of claim 1, wherein the vegetable oil is at least one of corn germ oil, rapeseed oil and cottonseed oil. The method for producing a heterologous protein according to claim 1, wherein the vegetable oil is added in a medium of 0.5 to 2%. The method for producing a heterologous protein according to claim 1, further comprising adding an antioxidant to the medium. 5. The method of claim 4, wherein the antioxidant is at least one of tocopherol and astaxanthin. The method for producing a heterologous protein according to claim 4, wherein the antioxidant is added in an amount of 0.01 to 0.1% in the medium. The method of claim 1, wherein the concentration of methanol in the medium is maintained at 0.5 to 4%. The method of claim 1, wherein the recombinant methanolized yeast is Hanshenula polymorpha DL1-008 (KCTC 8677P).