KR970006156B1 - A process for culturing of recombinated yeast containing a dna of presurface antigen and surface antigen in hepatitis type b virus - Google Patents

Abstract

In case that enzyme maternal cell including preS2-S Ag protein gene controlled by glyceroaldehyde-3-phosphoric dehydration enzyme(GAP) promoter is cultivated, recombinant yeast is produced by supplying and cultivating excess substrate to keep the concentration of ethanol over a certain standard since when the concentration of dextrose is 0. Therefore, the above cultivation method of recombinant yeast can improve the productivity of entire surface and surface antigen.

Description

Method for cultivating recombinant yeast containing genes of full surface antigen and surface antigen of non (B) hepatitis virus

1 is a diagram showing the increase in productivity per unit cell of BGUD hepatitis whole surface antigen and surface antigen by maintaining the ethanol concentration in the culture medium by more than 10g / L by the fed-batch culture of the present invention by the constant rate addition of YEPD medium .

Figure 2 is a fed-batch culture in which YEPD medium is supplied as much as the cell requires. The cell yield (Y) is 1.5 (0D ^* L / glucose) and the substrate excess supply variable (a) is 1 Figure 1 shows that the productivity per unit cell is drastically reduced when the medium is fed at a feed rate such that the specific growth rate of is controlled to 0.06 (/ hr).

Figure 3 is a fed-batch culture in which YEPD medium is supplied only as much as the cell requires. The cell yield (Y) is 1.5 (0D ^* L / glucose) and the substrate excess supply variable (a) is 1 Figure 1 shows that the productivity per unit cell decreases when the medium is fed at a feed rate that allows the specific growth rate to be adjusted to 0.12 (/ hr).

4 is a diagram showing that the productivity per unit cell was increased when the concentration of ethane in the culture medium after culture under the same conditions as in FIG. 3 when the addition of YEPETOH (yeast extract, yeast peptone, ethanol) medium before addition.

5 is a fed-batch culture in which the YEPD medium is supplied in excess of the amount required by the cells. The cell yield (Y) is 1.5 (0D ^* L / glucose) and the substrate excess supply variable (a) is 1.8. The total productivity of pre-s2-S Ag is more than 90% by high concentration cell culture, which maintains the maximum productivity per unit cell when the medium is supplied at a feed rate that controls the specific growth rate of the cells to 0.12 (/ hr). The figure shows the improvement.

* Explanation of symbols for main parts of the drawings

O.P. : Hop Intensity Glc: Glucose

EtOH: Ethanol

Sp-poly: preS2-S Ag production (mg / ∫10) per unit cell (0.D.10)

tot-poly: preS2-S Ag Total Production (mg / ∫) ER: Supply Rate

The present invention relates to a method for culturing recombinant yeast for improving the productivity of all surface and surface antigens of hepatitis B virus, and more particularly, hepatitis B linked to a GAP (glyceraldehyde-3-phosphate dehydrogenase) promoter. During fed-batch culture of yeast containing the surface antigen (HBsAg) gene of the virus (HBV) and the full surface antigen (preS2) gene, the surface by adding more than the required amount of substrate (hereinafter referred to as excess substrate) to maintain a constant ethanol concentration in the culture medium The present invention relates to a method for culturing recombinant yeast characterized by improving the productivity of antigens and whole surface antigens.

Factors affecting expression rate when foreign proteins are expressed using recombinant microorganisms8 are expression vectors, promoters, host cells, plasmid copy number, culture conditions (pH, temperature, dissolved oxygen concentration, medium). And culture methods (dilution, fed-batch, continuous, etc.), and the expression rate depends on the difference of each factor. In particular, when host cell and expression vector types are determined, proper control of culture conditions and methods is very important for improving protein productivity. As a method for obtaining a desired foreign protein with high yield using such recombinant microorganisms, various methods aimed at culturing high concentrations of cells are mainly used (Bitter et al., J. Med Virol. 25, 123). -140 (1988), Fieschko et al, Biotechnolo-gy and Bioengineering 25, 1113-1121 (1987)). After high concentration culture of the cells, the higher the expression level per unit cell, the higher the productivity of the foreign protein.

For high concentration culture of cells, fed-batch culture is used more than batch culture. A fed-batch culture method commonly used to express the surface antigen (HBsAg) of hepatitis B virus using recombinant yeast is the rate of consumption, cell concentration, and culture by cells of a carbon source (eg, glucose) used as a substrate. It is a method of supplying the required amount of substrate in proportion to the increase of cells while maintaining the specific growth rate of the cell based on the change in volume [Man-Bock Gu et al., Applied Microbiogy and Biotechnology 35, 46-50 (1991), Jih-Han Hsieh et al., Biotechnology and Bioengineering 32, 334-340 (1988).

However, by applying a conventional fed-batch culture that supplies only the amount of substrate required as described above, the genes of the pre-surface antigen and the surface antigen (preS2-S Ag) of the type B sensitized virus under the control of the GAP promoter are included. As a result of culturing yeast cells, it was possible to culture cells at high concentrations, but it was found that the overall productivity was still low due to the small amount of protein expressed per unit cell.

Accordingly, the present inventors have made a thorough study to increase the productivity per unit cell, and found that the production and consumption of ethane according to substrate supply correlated with the productivity per unit cell of preS2-S Ag. In other words, if the required amount of substrate is supplied, the ethanol concentration in the medium is close to zero, and high concentration culture of cells is realized, but the productivity per unit cell is low, whereas if the substrate is supplied in excess of the required amount, ethanol accumulates in the medium, but Productivity per cell is increased, and the cell yield lowered during substrate feeding due to overfeeding can be increased to the level that can be obtained from a culture in which the substrate is supplied as needed through a batch culture using ethanol remaining in the culture as a carbon source after the substrate feeding is finished. I found out.

Therefore, on the basis of this fact, the present inventors have found that it is difficult to cultivate high concentration of cells when ethanol accumulates, and thus, unlike the conventional fed-batch culture method of culturing by adjusting the ethanol concentration in the medium to almost 0, In addition, it is possible to invent a method for culturing yeast that increases productivity per unit cell.

Therefore, an object of the present invention is to supply a unit cell by supplying an excess substrate so that the ethanol concentration is maintained above a certain level from the time when the glucose concentration is 0 in the culture of yeast cells containing the gene of preS2-S Ag protein under the control of the GAP promoter It is to provide a method for culturing recombinant yeast that increases the productivity of sugar by increasing the sugar productivity.

Hereinafter, the present invention will be described.

During the substrate feeding period, an initial batch culture commonly used for fermentation of yeast cells is performed, followed by a fed-batch culture that controls the specific growth rate of cells while supplying excess substrate at a constant rate from the time when the glucose concentration becomes zero. By maintaining the ethanol concentration above a certain level it can be prevented to reduce the productivity per unit cells of preS2-S Ag. In this case, the cell yield lowered during substrate feeding due to oversupply is increased by continually growing cells using ethanol remaining in the culture as a carbon source during the batch culture after the substrate feeding is completed. It can be maintained at the level obtained from fed-batch cultures that provide as much as necessary.

The concentration of ethanol in the medium is preferably 10g / L or more, but when the ethanol concentration is 20g / L or more, the growth of cells is inhibited, so the ethanol concentration is most preferably in the range of 10-20g / L.

As the excess substrate, a medium containing a larger amount of glucose than that required for cell growth or a medium containing an amount of glucose and ethanol required for cell growth can be used.

Substrates can be added at a time after the end of the initial batch culture, continued to be fed at a rate specified for overfeeding, or added by a computer-controlled feeding method as described below. Feeding is most preferred.

In general, in high concentration culture of recombinant yeast, a flow rate determination formula such as Equation (1) is used to add YEPD medium in proportion to increasing cell concentration. Using the cell yield (Y) found in the batch fermentation and the specific growth rate (μ) of the cells to be controlled, the addition rate (F) is determined by the following equation.

In general, the value of a is 1, but the substrate is oversupplied at a constant rate using a value determined every minute by the computer to maintain the ethanol concentration above a certain level during the medium addition period. Almost but no ethanol concentration is maintained above 10 g / L, preferably between 10 and 20 g / L, it is advisable to maintain the substrate overfeed parameter a at least 1, preferably 1 to 2, most preferably 1.3 to 2.0. .

In addition, the specific growth rate of the cells is preferably maintained at 0.1 to 0.15 (/ hr).

In this manner, the fed-batch culture of oversubstrate can increase the productivity per unit cell of preS2-S Ag. This effect is always open to the GAP (glyceraldehyde-3-phosphate dehydrogenase) promoter used by the present inventors. In the light of the fact that it is a promoter and is stronger in the presence of glucose than ethanol, the protein expression is made by glucose during substrate feeding, so that the increase in productivity per unit cell in the presence of ethanol in the medium is greater than the time of expression, e.g. It is expected that ethanol has a significant effect on the particle formation of expressed proteins.

Hereinafter, the present invention will be described in more detail with reference to Examples. The following examples merely illustrate the invention and do not limit the scope of the invention.

In an embodiment of the present invention, the yeast Saccharomyces cerevis transformed with a vector (pYLBC-GAP-HBV) having a BGUD hepatitis virus surface antigen (HBsAg) gene, a full surface antigen (pre-S2) gene, and a yeast promoter GAP. Saccharomyces cerevisiae was used, but not limited thereto. This strain was deposited on December 14, 1993 under the Korean spawn accession no. KCTC 0098BP.

In the following examples and comparative examples, seed culture of the initial strain was performed as follows.

Strain 1 vials (1 ml) stored in a -80 ° C freezer were inoculated into 50 ml of leucine deficiency medium (yeast nitrogen base 0.67% adenine 0.004%, caged 0.003%, threonine 0.04%, glucose 6%) in a shake stirrer. After incubation for 24 hours at 30 ℃, 200rpm inoculated in YEPD medium (1% yeast extract, 2% yeast peptone, 8% glucose) at 5% (v / v) ratio and 30 ℃ in shaking stirrer for 12 to 15 hours And incubated at 200 rpm.

Protein quantification was quantified using polyclonal antibodies obtained by injecting rabbits with a type B infection surface antigen commonly used as an international standard purchased from NSBS under the World Health Organization (WHO).

Example 1

A constant rate feed organic culture method for feeding a substrate so that the concentration of ethane is above a certain level.

Feeding rate of the addition medium so that the ethanol concentration is above a certain level during the medium addition section in the fed-batch culture so that the productivity per unit cell is increased for high concentration culture of the recombinant yeast producing the full surface antigen and the surface antigen of the hepatitis B virus. Fed-batch cul-ture by constant addition rate was carried out to keep the constant.

The seed culture solution was inoculated in 2.2 L of the initial YEPD medium (3% yeast extract 1%, glucose 2%) to 5% (v / v) ratio to pH 4.5-5.5,27 ℃, 500-900rpm, 1vvm After the batch culture was started, the final 3L volume of YEPD medium was prepared to have 6% YEPD (yeast extract), 3% P (east peptone), and 6% D (glucose). Medium was fed at an addition rate of 1,25 ml / min. After about 10 hours of addition, as shown in Figure 1, the ethanol concentration was continuously supplied in the range of 10-20 g / L during the addition period, and the productivity per unit cell of preS2-S Ag protein was increased. Continued to increase.

Comparative Example 1

Oil culture to control the cell's specific growth rate to 0.06 / hr for high concentration cell culture that supplies only the amount of substrate required by the cell

In order to cultivate the cells at a high concentration, a fed-batch culture was performed as follows.

The seed culture solution was inoculated to 2.2% of the initial YEPD medium (3% yeast extract, 1% yeast peptone) at a rate of 5% (v / v) and subjected to batch culture at a pH of 4.5-5.5,27 ° C and 500-900 rpm 1vvm aeration rate. Started. In this case, since glucose was not included in the initial medium, glucose was randomly made to a time point of zero. Therefore, the supplemented medium prepared so that the composition of the final 3L volume of YEPD medium was YE 6%, P3%, D 6% from the beginning of the depletion of all the glucose in the culture medium was 1, and the cell yield (Y) Is 1.5. Substitute the cell concentration (OD) and the culture volume (V) measured at the time of addition, and add at the substrate feed rate determined by the substrate feed rate determination formula (1) so that the specific growth rate of the cells is 0.06.hr. It was. As a result, as shown in FIG. 2, the ethanol concentration in the culture medium started to decrease from the time when the substrate was added, and maintained at 0 after 15 hours.

When the substrate was supplied in the amount required by the cells in proportion to the increase in the cell concentration, high concentration cell culture with a final cell concentration of OD 120 was possible. However, at this time, the productivity per unit cell (OD 10) of the hepatitis B whole surface antigen and the surface antigen protein was continuously decreased, and thus the total productivity was lower than that of Example 1. It is thought that this is because the specific growth rate of the cells is very low, such as 0.06 / hr, so that the second half of the medium addition promotes the degradation of the entire surface antigens and surface antigen proteins of the hepatitis B virus produced by the action of proteolytic enzymes in the cells. Therefore, in the case of fed-batch culture at a specific growth rate of 0.06 / hr, high concentration of cells is possible, but due to the rapid decrease in productivity per unit cell, the overall productivity cannot be increased as the cell concentration increases.

Comparative Example 2

Feeding value culture to control the cell's specific growth rate by 0.12 / hr for high-density cell culture that supplies only the amount of substrate required by the cell

As in Comparative Example 1, the low growth rate of the cells was very low, and a fed-batch culture was performed to adjust the specific growth rate of the cells to 0.12.hr to prevent the possibility of proteolysis.

The seed culture solution was inoculated to 2.2% of the initial YEPD medium (3% yeast extract, 1% yeast peptone, 2% glucose) at a rate of 5% (v / v) and aeration at pH 4.5-5.5,27 ℃, 500-900rpm, 1vvm When the batch culture was started at the rate and the glucose in the culture was depleted, the addition medium prepared so that the composition of the final 3L volume of the YEPD medium was 6%, P3%, and D6% of YE was overfeed variable a and the cell yield was 1. (Y) is set at 1.5 to supply the interval substrate by the substrate feed rate determination formula (1) by substituting the cell concentration (OD) and the culture volume (V) measured at the time of addition so that the specific growth rate of the cells is 0.12 / hr. It was added at a rate. As a result, as shown in FIG. 3, the final cell yield was somewhat decreased compared to the case where the final cell concentration was OD 80-90 at 0.06 / hr, but high concentration cell culture was possible. And since the addition per unit cell productivity does not tend to decrease sharply as in the case of Comparative Example 1 after the addition, the constant-rate fed-value fed culture of Example 1 showed a lower value than the result of the fed-batch culture.

Example 2.

Cultivation by the addition of fed-batch YEPEtOH medium.

To investigate the effect of ethanol concentration on the productivity per unit cell of preS2-S Ag protein in fed-batch culture, the following culture method was used.

First, in the fed-batch culture method, after adding the substrate under the same conditions and methods as in Comparative Example 2, YEPHtOH medium (20% yeast extract, 6% yeast peptone 12% ethanol) was added at a time when the ethanol concentration in the culture medium was 0. When added, preS2-S Ag showed higher productivity per cell than before YEPEtOH medium, and the growth of cells using ethanol as carbon source continued, resulting in high concentration cell culture with final cell concentration of OD 75 or more. It was.

In the case of Comparative Example 2 (see FIG. 3), the productivity per unit cell decreases when the substrate is supplied by the feeding formula even after the ethanol concentration in the culture solution becomes zero. The addition of the included medium was overcome by adjusting the concentration of ethanol in the culture. This result shows that while protein expression is performed by glucose during substrate feeding by the action of the GAP promoter, ethanol has an important effect on the particle formation site of the expressed protein. It is thought that the particle formation rate of preS2-S Ag protein increased.

Example 3

Determination of fed-batch cultivation method that oversubscribes the substrate to the amount needed by the cells

Reduction of ethanol concentration during the fed-batch culture for high-density cell cultivation in case of obtaining preS2-SAg protein using recombinant yeast including GAP (glyceraldehyde-3 phosphate dehydrogenase) promoter reduces preS2-S Ag productivity per unit cell It was found in the comparative example that the cause of the above, and the results were synthesized to devise a method of maintaining the ethanol concentration of 10g / L or more by supplying the substrate in excess of the amount required by the cell according to the following formula.

The seed culture solution was inoculated at a rate of 5% (v / v) in 2.2 L of the initial YEPD (3% yeast extract, 1% yeast peptone, 2% glucose) and batched at a pH of 4.5-5.5, 27 ° C. and 500-900 rpm. After the start of the culture, the addition medium prepared so that the composition of the final 3L volume of YEPD medium was YE 6%, P 3%, and D6% from the time when all the grape dies were depleted in the culture medium was oversupplied by using a substrate feeding rate determination formula . In other words, in the formula (1), the substrate excess supply variable (a) was 1.8 and the cell yield (Y) was 1.5. Substituting the cell concentration (OD) and the culture volume (V) measured at the time of addition, the specific growth rate of the cells was substituted. Was adjusted to 0.12 / hr.

As a result, as shown in FIG. 5, the ethanol concentration was maintained in the range of 10-20 g / L during the substrate feeding section, and the preS2-S Ag productivity per unit cell could be maintained or increased. On the other hand, after the substrate supply was completed, cell growth using the ethanol remaining in the culture as a carbon source was continued, and the total cell concentration (OD) reached about 80, resulting in high concentration cell culture maintaining maximum productivity per unit cell. Overall productivity increased by more than 90% compared to no oversupply.

Claims (6)

A meat culture method of the manufactured yeast transformed by the surface antigen (HBsAg) gene and the whole surface antigen (pres2) gene of hepatitis B virus (HBV) under the control of the GAP (glyceraldehyde-3-phosphate dehydrogenase) promoter The method of culturing recombinant yeast, characterized in that an excess substrate is added so that the ethanol concentration is maintained at a predetermined level or more from the time when the glucose concentration in the culture medium becomes zero. The method of claim 1 wherein the ethanol concentration is maintained in the range of 10 to 20 g / L. The process according to claim 1 or 2, wherein the excess substrate is added at a feed rate obtained by the following formula. The method of claim 3, wherein the substrate overfeed parameter is between 1 and 2 and the cell specific growth rate is between 0.10 and 0.15 (/ hr). The method of claim 4, wherein the substrate overfeed parameter is between 1.3 and 2.0. The method of claim 1, wherein the recombinant yeast is Saccharomyces cerevisiae (Accession Number: KCTC 0098BP).