KR100241266B1 - Process for improving productivity of human alpha-iterferon from recombinant yeast - Google Patents

Abstract

The present invention relates to a method for mass production of human alpha-interferon (IFN-α) in high yield by controlling the pH of the fermentation medium in a recombinant yeast strain. Specifically, the present invention provides a recombinant yeast expressing and secreting IFN-α under the control of the ADH2 / GAP promoter. 1) maintaining the pH of the fermentation medium higher than the optimal pH for yeast growth for the entire fermentation process, or 2) yeast. The present invention relates to a method of significantly increasing the yield of IFN-α in the recombinant yeast strain by fermenting at an optimal growth pH and maintaining the pH of the fermentation medium in the IFN-α secretion step.

Description

How to produce human alpha-interferon in high yield in recombinant yeast

The present invention relates to a method for mass production of human alpha-interferon (IFN-α) in high yield by controlling the pH of the fermentation medium in a recombinant yeast strain.

More specifically, the present invention provides a recombinant yeast expressing and secreting IFN-α under the control of the ADH2 / GAP promoter, 1) maintaining the pH of the fermentation medium higher than the pH optimal for yeast growth during the entire fermentation process; The present invention relates to a method of greatly increasing the yield of IFN-α in the recombinant yeast strain by fermenting the yeast at a growth optimum pH and then maintaining the pH of the fermentation medium in the IFN-α secretion step.

After human insulin is produced from recombinant E. coli using genetic engineering techniques, many useful pharmaceutical proteins such as interferon (IFN), hepatitis vaccine and human growth hormone are produced in large quantities in microorganisms. It is marketed. However, since genetic engineering techniques have a low yield of fermentation and production of pharmaceutical proteins in microorganisms, these methods have been mainly used to produce expensive pharmaceutical proteins or enzymes.

Specifically, the production process of a pharmaceutical protein using microorganisms consists of a multi-step process of first developing a microorganism capable of producing the protein by genetic engineering method, producing the protein by fermentation process, and finally separating and purifying the product protein. It is. Therefore, studies to improve the production yield of the pharmaceutical protein by improving the fermentation process of the microorganism in order to improve the productivity has been continued, and the research on the recombinant pharmaceutical protein is increasingly accelerated.

To improve the fermentation productivity of the microorganism, first, the expression vector at the gene level can be manipulated to increase the fermentation efficiency of the recombinant gene. Since the expression rate of the recombinant gene and the growth rate of the cell are inversely related to each other, the product protein can be obtained with high yield by separating the cell growth time and the product protein expression time. In order to form a condition in which cell growth time and product protein expression time are separated as described above, a regulated promoter is generally used as a genetic switch, which is an environmental factor such as temperature, nutrients and inducers ( inducer), etc., effectively induces protein expression, so it is easy to separate cell growth and product protein. For example, the ADH2 / GAP (alcohol dehydrogenase 2 / glyceraldehyde-3-phosphate dehydrogenase) promoter used in recombinant yeast is a product of yeast growth time and products. It provides a genetic system capable of separating the expression alc secretion time of the protein.

The yield of protein can then be increased by fed-batch culture or two-stage continuous culture at the reactor level. Specifically, in the early stage of fermentation, the growth rate of the cells is maintained to the maximum, and the concentration of the cells is increased in the shortest time, and then the genes encoding the product proteins are expressed. Can be.

Therefore, fermentation with optimum fermentation conditions for each recombinant microorganism and product protein is important for mass production of the product protein. For example, it has been reported that productivity is increased fourfold by optimizing the expression of amylase, a molecular oligosaccharide producing enzyme, by optimizing the medium composition (Nam Seung-heon, Korea Food Science Society, Bacillus licheniformis amylase) 26: 411-416 (1994)).

IFN-α is a protein with antiviral activity secreted from cells infected with viruses or viral RNA, and many studies have been conducted since the first IFN-α gene was isolated in the early 1980s. Since the antiviral activity of IFN-α can be usefully used to treat viral diseases, many methods for producing IFN-α in microorganisms have been developed to genetically mass-produce IFN-α.

Yeast is a strain useful for expressing foreign proteins derived from eukaryotic cells. When yeast ferments foreign proteins outside of yeast cells, it is easier to purify proteins than they are expressed in cells. (Masakasu Kiduchi and Morio Ikehara, TIBTECH, “Conformational features of signal sequences and folding of secretory proteins in yeasts”, 208-211 (1991. 6)) .

Specifically, we can produce IFN-α in yeast, including the ADH2 / GAP promoter, the α-factor leader sequence of yeast, and the gene of IFN-α to produce IFN-α in yeast. Recombinant plasmids were prepared and transformed into yeast strains to obtain recombinant yeast Saccharomyces cerevisiae DCO4 strain (Korean Patent Application No. 92-16766, Accession No .: KCTC 0051 BP), from which IFN-α was produced.

The expression promoter ADH2 / GAP promoter used in the recombinant plasmid has the property of inhibiting expression when glucose is present in the medium and starting expression when glucose is depleted, and the foreign protein expressed therefrom is expressed by the α-factor leading sequence of the yeast. When yeast germinates and divides, it is secreted. In order to accumulate desired recombinant protein in high concentration in the fermentation medium, the expression by the ADH2 / GAP promoter and the secretion by the α-factor leading sequence of the yeast are preferably maintained so that glucose is depleted in the fermentation medium and expression starts. from the time that a specific growth rate of yeast appropriate level (0.08-0.12hr ^-) the addition rate of glucose release constantly supplied from the time that the fermentation process starts to be maintained, to be controlled exponentially. In this case, glucose and ethanol should not accumulate in the medium by using glucose completely in the yeast (see Korean Patent Application No. 94-22289).

However, when the yeast producing IFN-α is expressed in a general manner, only about 50% of the IFN-α produced is folded, and the remaining unfolded IFN-α is easily separated by conventional purification processes. There was a problem that the yield is sharply reduced even if not separated. The degree of folding of the protein depends on the type of protein expressed, but if it is not folded and is exposed to the fermentor for a long time, the protein may be degraded or denatured. In particular, proteins may be entangled in the process of concentrating the fermentation broth to start purification Biological activity is reduced. In general, an oxidation process using metal ions (Journal of Biological Chemistry, 258 (19), 11834-11830) is used to fold proteins during separation and purification.

Using the above facts, when the yeast strain transformed with the IFN-α gene is grown in a fed-batch in fermentation medium containing metal ions to produce IFN-α, the folding of the secreted IFN-α is promoted. Yield was significantly improved (Korean Patent Application No. 96-26844). In addition, it was confirmed that even if only copper ions were added to the synthesis medium in the above production method, the folding of IFN-α was promoted and the yield was improved (see Korean Patent Application No. 97-9188). However, in the above invention, IFN-α is mass-produced by fed-batch, and fed-batch culture can obtain higher yeast cell concentration than batch culture, but the amount of IFN-α secreted is not relatively high compared to high cell concentration. There is a problem that this complex and costs a lot of raw materials.

On the other hand, when the starch-fermenting yeast starch-fermenting yeast, Schwanniomyces alluvius produces the secreted protein amylase, the enzyme is secreted at a pH higher than the optimum pH of the yeast fermentation medium. It has been reported that shortening of residence time in yeast periplasm causes the secretion of protein (GB Calleja et al., Critical Reviews in Biotechnology, volume 5, issue 3, 177-184 (1987). )).

In order to mass produce IFN-α in a high yield in recombinant yeast Saccharomyces cerevisiae, the present inventors maintained the recombinant yeast strain higher than the pH optimum for yeast growth for the entire fermentation process, or the yeast was fermented at a growth optimum pH. The present invention was completed by increasing the production of IFN-α in the process of maintaining a high pH of the fermentation medium in the next protein secretion step.

It is an object of the present invention to provide a method for greatly increasing the production of IFN-α by controlling the pH of a fermentation medium in a recombinant yeast strain.

1 is a result of confirming non-reducible SDS-polyacrylamide gel electrophoresis of the production of human alpha-interferon which varies with pH when batch culture of the recombinant yeast strain while controlling the pH of the fermentation medium by the method of the present invention. .

In order to achieve the above object, the present invention provides a method for producing IFN-α in high yield by maintaining the pH of the fermentation medium of the yeast production of the fermentation medium higher than the pH optimum for yeast growth for the entire period of the fermentation process to provide. In addition, the present invention provides a method for producing IFN-α in high yield by fermenting recombinant yeast at an optimal growth pH of yeast and then maintaining the pH of the fermentation medium high from the IFN-α secretion stage.

Hereinafter, the present invention will be described in detail.

The present invention provides a method of increasing the production of IFN-α by adjusting the pH of the fermentation medium in fermentation to produce IFN-α from a recombinant yeast strain.

The present invention provides a recombinant plasmid capable of producing IFN-α in yeast, including the ADH2 / GAP promoter, the α-factor leader sequence of the yeast, and the IFN-α gene in order to mass produce IFN-α in high yield in yeast. The recombinant yeast Saccharomyces cerevisiae DCO4 strain obtained by transforming the strain is used (Korean Patent Application No. 92-16766; Accession No .: KCTC 0051 BP).

Specifically, the present invention increases the yield of INF-α by maintaining a recombinant yeast strain producing IFN-α higher than 5.0, which is an optimal pH for yeast growth throughout the fermentation process, wherein the pH of the fermentation medium is It is preferable to keep it in the range of 6.0 to 7.5 (see FIG. 1 and Table 1).

In another aspect, the present invention provides a two-step method for maintaining the pH of the fermentation medium in the cell growth step of the recombinant yeast strain to produce the optimum pH, IFN-α secretion step to maintain a high pH. In adjusting the pH in two stages as described above, the time point of changing the pH is most preferably the time when the expression of IFN-α is depleted due to the depletion of glucose. At this time, the yeast is fermented at a growth optimum pH of 5.0, then in the IFN-α secretion step it is preferable to maintain the pH of the fermentation medium in the range of 6.0 to 7.5 (see Table 4).

The method of the present invention can be used in all cases where the recombinant yeast strain producing IFN-α is cultured batchwise or fed-batch.

As a result of expressing IFN-α by the method of the present invention, the concentration and titer of IFN-α were measured by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and cytopathy. It was confirmed that as the production of IFN-α is greatly increased (see FIG. 1, Table 1 and Table 4).

When the recombinant yeast is fermented batchwise at pH 6.0-7.5, the cell growth is slightly decreased compared to when yeast is fermented at the optimum pH of yeast 5.0, but the production of IFN-α secreted into the medium is at least 2 to 10 times. Is increased. In addition, the fermentation of recombinant yeast by batch batch fermentation at high pH is more than four times higher than when fermenting at pH 5.0 in complex fed-batch, and the pH is changed to 5.0 when fermented by changing the pH by two-stage fed-batch. The production of IFN-α is increased up to four times more than the fed-batch that is maintained and fermented.

Hereinafter, the present invention will be described in detail.

However, the following examples are merely to illustrate the invention, the present invention is not limited by the examples.

Comparative Example 1

[Batch fermentation with pH of fermentation medium adjusted to 5.0]

0.4 ml of Saccharomyces cerevisiae DCO4 strain (Accession No .: KCTC 0051 BP), which is transformed to secrete and produce IFN-α, and 150 ml of YPD medium (10 g / L yeast extract, 20 g / L yeast peptone, glucose) 80 g / L) and fermented at 30 ° C. at a stirring speed of 200 rpm for 16 hours in a shaker, and then the seed culture medium was added to YPD medium (10 g / L yeast, 20 g / L yeast peptone, 10 g / L glucose). Inoculated at a rate of% (v / v) in a fermenter at agitation rate at 30 ° C 500rpm aeration rate was fermented for 72 hours at 1vvm. At this time, the pH was adjusted to 5.0 using ammonia water from the time of the main culture inoculation, and the error of pH was ± 0.1.

FIG. 1 is a result of confirming the amount of IFN-α by non-reducing SDS-polyacrylamide gel electrophoresis. All samples were collected after 31 hours of inoculation with the seed culture broth. The upper part of FIG. 1 is the grooved side of IFN-α loaded with SDS in the polyacrylamide gel, wherein the reduced IFN-α (Red) is moved from the oxidized IFN-α (Ox). It appears to be slow. However, the reduced form of IFN-α exhibits similar activity to that of the oxidized form, so the sum of the oxidized and reduced forms is the total amount of IFN-α produced. Where column M is the molecular weight label of the standard protein and column 2 is the amount of IFN-α produced when the pH of the fermentation medium is 5.0. Table 1 shows the titer of IFN-α analyzed by cytopathic method. When the pH of the fermentation medium is 5.0, the titer of IFN-α is 1.8 × 10 ⁷ IU / ml.

Example 1

[Batch fermentation with pH of fermentation medium adjusted to 4.5]

0.4 ml of Saccharomyces cerevisiae DCO4 strain (Accession No .: KCTC 0051 BP), transformed to secrete and produce IFN-α, and 150 ml of YPD medium (10 g / L yeast extract, 20 g / L yeast peptone, 80 g glucose) / L) and fermented at 30 ° C. at a stirring speed of 200 rpm for 16 hours in a shaker, followed by culturing the seed culture solution in YPD medium (yeast extract 10g / L, yeast peptone 20g / L, glucose 10g / L). Inoculated at a rate of (v / v) and fermented at a stirring speed of 500 rpm at 30 ° C. in a fermenter at 1 vvm for 72 hours. At this time, the pH was adjusted to 4.5 using ammonia water from the time of the main culture inoculation, and the error of pH was ± 0.1. Column 1 of FIG. 1 is the amount of IFN-α produced when the pH of the fermentation medium is 4.5. As shown in Table 1, when the pH of the fermentation medium is 4.5, the titer of IFN-α is 4.8 × 10 ⁶ IU / ml. As shown in FIG. 1 and Table 1, the production of IFN-α was lower when the pH of Comparative Example 1 was maintained at pH 4.5 lower than the pH optimal for yeast growth than that of 5.0.

Example 2

[Batch Fermentation Adjusting pH of Fermentation Medium to 6.0]

0.4 ml of Saccharomyces cerevisiae DCO4 strain (Accession No .: KCTC 0051 BP), transformed to secrete and produce IFN-α, and 150 ml of YPD medium (10 g / L yeast extract, 20 g / L yeast peptone, 80 g glucose) / L) and fermented at 30 ° C. at a stirring speed of 200 rpm for 16 hours in a shaker, followed by culturing the seed culture solution in YPD medium (yeast extract 10g / L, yeast peptone 20g / L, glucose 10g / L). Inoculated at a rate of (v / v) and fermented at a stirring speed of 500 rpm at 30 ° C. in a fermenter at 1 vvm for 72 hours. At this time, the pH was adjusted to 6.0 using ammonia water from the time of the main culture inoculation, and the error of pH was ± 0.1. Row 3 of FIG. 1 shows the amount of IFN-α produced when the pH of the fermentation medium of Example 2 is 6.0. As shown in Table 1, when the pH of the fermentation medium is 6.0, the titer of IFN-α is 3.6 × 10 ⁷ IU / ml. As can be seen from FIG. 1 and Table 1, the production of IFN-α was twice higher when the pH of Comparative Example 1 was maintained at pH 6.0 higher than the pH optimum for yeast growth than that of 5.0.

Example 3

[Batch fermentation with pH of fermentation medium adjusted to 6.5]

0.4 ml of Saccharomyces cerevisiae DCO4 strain (Accession No .: KCTC 0051 BP), transformed to secrete and produce IFN-α, and 150 ml of YPD medium (10 g / L yeast extract, 20 g / L yeast peptone, 80 g glucose) / L) and fermented at 30 ° C. at a stirring speed of 200 rpm for 16 hours in a shaker, followed by culturing the seed culture solution in YPD medium (yeast extract 10g / L, yeast peptone 20g / L, glucose 10g / L). Inoculated at a rate of (v / v) and fermented at a stirring speed of 500 rpm at 30 ° C. in a fermenter at 1 vvm for 72 hours. At this time, the pH was adjusted to 6.5 using ammonia water from the time of the main culture inoculation, and the error of pH was ± 0.1. Row 4 of FIG. 1 shows the amount of IFN-α produced when the pH of the fermentation medium of Example 3 is 6.5. As shown in Table 1, when the pH of the fermentation medium is 6.5, the titer of IFN-α is 7.2 × 10 ⁷ IU / ml. As shown in FIG. 1 and Table 1, the production of IFN-α was 4 times higher when the pH of Comparative Example 1 was maintained at pH 6.5 lower than the pH optimum for yeast growth than that of 5.0.

Example 4

[Batch fermentation with pH of fermentation medium adjusted to 7.0]

0.4 ml of Saccharomyces cerevisiae DCO4 strain (Accession No .: KCTC 0051 BP), transformed to secrete and produce IFN-α, and 150 ml of YPD medium (10 g / L yeast extract, 20 g / L yeast peptone, 80 g glucose) / L) and fermented at 30 ° C. at a stirring speed of 200 rpm for 16 hours in a shaker, followed by culturing the seed culture solution in YPD medium (yeast extract 10g / L, yeast peptone 20g / L, glucose 10g / L). Inoculated at a rate of (v / v) and fermented at a stirring speed of 500 rpm at 30 ° C. in a fermenter at 1 vvm for 72 hours. At this time, the pH was adjusted to 7.0 using ammonia water from the time of the main culture inoculation, and the error of pH was ± 0.1. Column 5 of FIG. 1 is the amount of IFN-α produced when the pH of the fermentation medium of Example 4 is 7.0. As shown in Table 1, when the pH of the fermentation medium is 7.0, the titer of IFN-α is 7.8 × 10 ⁶ IU / ml. As shown in FIG. 1 and Table 1, the production of IFN-α was 4.3 times higher when the pH of Comparative Example 1 was maintained at pH 7.0 lower than the pH optimum for yeast growth, compared to pH 5.0.

Example 5

[Batch fermentation with pH of fermentation medium adjusted to 7.5]

0.4 ml of Saccharomyces cerevisiae DCO4 strain (Accession No .: KCTC 0051 BP), transformed to secrete and produce IFN-α, and 150 ml of YPD medium (10 g / L yeast extract, 20 g / L yeast peptone, 80 g glucose) / L) and fermented at 30 ° C. at a stirring speed of 200 rpm for 16 hours in a shaker, followed by culturing the seed culture solution in YPD medium (yeast extract 10g / L, yeast peptone 20g / L, glucose 10g / L). Inoculated at a rate of (v / v) and fermented at a stirring speed of 500 rpm at 30 ° C. in a fermenter at 1 vvm for 72 hours. At this time, the pH was adjusted to 7.5 using ammonia water from the time of the main culture inoculation, and the error of pH was ± 0.1. Row 6 of FIG. 1 shows the amount of IFN-α produced when the pH of the fermentation medium of Example 5 is 7.5. As shown in Table 1, when the pH of the fermentation medium is 5.0, the titer of IFN-α is 3.7 × 10 ⁷ IU / ml. As shown in FIG. 1 and Table 1, the production of IFN-α was twice higher when the pH of Comparative Example 1 was maintained at pH 7.5 lower than the pH optimum for yeast growth than that of 5.0.

As shown in the above example, when the pH was 4.5, the lowest amount of IFN-α was shown, and as the pH was increased, the amount of IFN-α was increased. As a result, it can be seen that the amount of IFN-α produced is greatly increased when the pH is adjusted to pH 6.0 to 7.5 higher than 5.0 known as the most optimal pH for yeast.

Example 6

[Verification of IFM-α by Cytopathy]

In the present invention, the titer of IFN-α was more sensitively confirmed by cytopathy, a biological assay for testing antiviral activity against cells. The antiviral activity against vesicular Stomatitis Virus (ATCC VR 158) infected with MDBK (ATCC CCL 32), which is a bovine kidney cell, is determined to confirm the activity of the IFN-α. The results of the cytopathology used were up to 50% error.

The seed cultures were inoculated in the main culture, fermented for 31 hours, and then the IFN-α titers of the collected samples were confirmed by cytopathy. As shown in Table 1, when the pH of the fermentation medium is 7.0, it shows the highest titer of IFN-α, which is 4.3 times higher than the case where the pH of the fermentation medium is 5.0, which is optimal for yeast growth. .

Example 7

[Batch fermentation using a medium in which the pH of the fermentation medium is adjusted to 7.0 and the concentration of glucose is changed]

0.4 ml of Saccharomyces cerevisiae DCO4 strain (Accession No .: KCTC 0051 BP), transformed to secrete and produce IFN-α, and 150 ml of YPD medium (10 g / L yeast extract, 20 g / L yeast peptone, 80 g glucose) / L) and fermented for 16 hours at a stirring speed of 200rpm at 30 ℃ in a shaker in a shaker, and then the culture medium was YPD medium with a glucose concentration of 1 to 5% (yeast extract 10g / L, yeast peptone 20g / L, 10-50 g / L) glucose was inoculated at a rate of 5% (v / v) and fermented at 30 ° C. in a fermenter at agitation speed of 500 rpm for 1 hour at 1 vmv for 72 hours. At this time, the pH was adjusted to 7.0 using ammonia water from the time of the main culture inoculation, and the error of pH was ± 0.1. As a result, when the concentration of glucose in the fermentation medium was 2%, it was confirmed that IFN-α production was the highest at 1.3 × 10 ⁸ IU / ml.

Example 8

[Batch fermentation in which the pH of the fermentation medium was adjusted in two stages]

0.4 ml of Saccharomyces cerevisiae DCO4 strain (Accession No .: KCTC 0051 BP), transformed to secrete and produce IFN-α, and 150 ml of YPD medium (10 g / L yeast extract, 20 g / L yeast peptone, 80 g glucose) / L) and fermented for 16 hours at agitation speed 200rpm at 30 ℃ in a shaker, and then the seed culture broth was YPD medium (10g / L yeast extract, 20g / L yeast peptone, 10g glucose) with a glucose concentration of 2% / L) was inoculated at a rate of 5% (v / v) and fermented at a stirring rate of 500 rpm at 30 ° C. at a fermentation rate of 1 vmv for 72 hours. At this time, the pH was maintained at 5.0 using ammonia water from the time of inoculation, and the pH was adjusted to 7.0 immediately after glucose depletion, and the error of pH was ± 0.1.

The results of Example 7, the fermentation with a pH of 7.0 with the highest yield and Example 8, fermented in two stages, were compared with the cytopathic method. Example 8 was 40% higher than IFN-α. It can be seen that the titer.

Comparative Example 2

[Federated Fermentation with pH of Fermentation Medium Adjusted to 5.0]

After culturing in the same manner as in Comparative Example 1, the secondary cultivation (fermentation volume 0.7 L) in 8% YEPD medium using 16.6 ml of the seed culture solution and inoculated in 8.3 L of the fermentation start medium, and then, in Table 2 and Table 3 2.7 L of the addition medium having the composition was added at a constant rate (1 L / hr) and fermentation (15 L fermenter, Keymap, Switzerland) was started. The rate of addition began to increase exponentially from the point where the ethanol in the medium was depleted. Dissolved oxygen (pO ₂ ) in the medium was maintained at 10% or more through agitation speed, aeration rate, pressure control in the fermentor and pH was maintained at 5.0 throughout the fermentation process. Table 4 shows the titers of IFN-α, and in this comparative example, the titers of IFN-α were 3.2 × 10 ⁷ IU / ml.

Example 9

[Fed-Batch Fermentation by Adjusting pH of Fermentation Medium in Two Stages of 5.0 and 6.0]

After culturing in the same manner as in Comparative Example 1, the secondary cultivation (fermentation volume 0.7 L) in 8% YEPD medium using 16.6 ml of the seed culture solution and inoculated in 8.3 L of the fermentation start medium, and then, in Table 2 and Table 3 2.7 L of the addition medium having the composition was added at a constant rate (1 L / hr) and fermentation (15 L fermenter, Keymap, Switzerland) was started. The pH of the fermentation medium was maintained at 5.0, which is optimal for yeast cell growth, and then began to increase exponentially the rate of addition while maintaining the pH at 6.0 at the point when the ethanol was depleted in the medium. Dissolved oxygen (pO ₂ ) in the medium was maintained at 10% or more through agitation rate, aeration rate, pressure control in the fermentor. Table 4 shows the titers of IFN-α, and the method of adjusting the pH in two stages as in the case of the present Example compared to the case in which the pH of the fermentation medium of Comparative Example 2 was maintained at 5.0 was 4 times the productivity of IFN-α. Higher.

Example 10

[Federated Fermentation Adjusting pH of Fermentation Medium to 5.0 and 7.0]

After culturing in the same manner as in Comparative Example 1, the secondary cultivation (0.7 L fermentation volume) in 8% YEPD medium using 16.6 ml of the seed culture solution, inoculated in 8.3 L of the fermentation start medium, and then added 2.7 L of the medium. Fermentation (15 L fermenter, Keymap, Switzerland) was started with addition at rate (1 L / hr). The pH of the fermentation medium was maintained at 5.0, which is optimal for yeast cell growth. From the point of depletion of ethanol in the medium, the rate of addition began to increase exponentially, keeping the pH high at 7.0. Dissolved oxygen (pO ₂ ) in the medium was maintained at 10% or more through agitation rate, aeration rate, pressure control in the fermentor.

As shown in Table 4, the pH of the fermentation medium of Comparative Example 2 was maintained at 5.0, and the method of adjusting the pH in two stages was 2.2 times higher in the productivity of IFN-α.

As described above, when the recombinant yeast was fermented in a batch range of pH 6.0-7.5, IFN-α production was increased by 4.3 times compared to the case where the fermentation was carried out at pH 5.0, and when the glucose concentration was 2% and fermented at high pH. Was increased by 7.2 times and was increased up to 10 times when fermentation pH was adjusted in two stages. In addition, the production by simply maintaining a high pH in a batch type is up to four times more than the case of a complex fed-batch culture with a pH of 5.0. The two-stage fed-batch culture method, in which yeast is grown at an optimum pH and then maintained at a high pH from the protein secretion step, increases the yield up to four times or more than the conventional fed-batch culture method.

Therefore, the method of the present invention produces IFN-α at a high yield of at least 2 to 10 times higher than if fermented at pH 5.0 by fermenting the recombinant yeast strain at a pH higher than 5.0, which is an optimal pH for yeast growth. Very useful for mass production of -α in high yield.

Claims (3)

During the fermentation process to produce human IFN-α from recombinant yeast strain Saccharomyces cerevisiae DCO4 (Accession No .: KCTC 0051 BP), IFN-α was maintained by maintaining the pH of the fermentation medium at pH 6.0-7.5. Manufacturing method to increase the yield of. The method of claim 1, comprising fermenting the recombinant yeast strain at a growth optimum pH, maintaining the pH of the fermentation medium at 6.0-7.5 in the IFN-α secretion step. The production method according to claim 1 or 2, wherein the recombinant yeast strain is fermented batchwise or fed-batch.

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