KR100251284B1 - How to improve the expression of granulocyte colony stimulating factor by two-step repeated feeding culture - Google Patents

Abstract

PURPOSE: Provided is a method for enhancing the expression of granulocyte colony stimulating factor, G-CSF, by two-stage repeated fed-batch process. Thereby, cell density, the stability of a plasmid and the expression level of G-CSF are stably maintained in high level at each fermenter, thus the productivity of G-CSF is enhanced if feeding fermentation is carried out repeatedly in cycle upto n times. CONSTITUTION: The expression of granulocyte colony stimulating factor, G-CSF, is enhanced by two-stage repeated fed-batch process while cultivating recombinant E.coli transformed with an expression vector of G-CSF using L-arabinose operon. The method is composed of the following steps of: (a) performing exponential feeding to control cell specific growth rate to 0.13h-1 - 0.18 h-1 and if cell density becomes 45g/L to 60g/L, transferring a part of fermentation fluid of the first stage into a fermenter of the second stage and growing the cell continuously in the remaining fermentation fluid by exponential feeding such that cell specific growth rate and cell density are constantly maintained; and (b) constant feeding at early dilution rate of 0.050h-1 - 0.120h-1 in a medium including glycerol as C source, to produce proteins after inducing protein expression by L-arabinose of the transferred cell.

Description

How to improve the expression of granulocyte colony stimulating factor by two-step repeated feeding culture

The present invention relates to a method for improving the expression of granulocyte colony stimulating factor (G-CSF) by a two-stage repeated fed-batch process. More specifically, the present invention relates to recombinant E. coli transformed with a G-CSF expression vector using L-arabinose operon of Salmonella strains. The present invention relates to a method of increasing the expression of recombinant G-CSF from E. coli by performing repeated fed-culture in steps.

In general, in fed-batch fermentation, the growth rate of the cells is proportional to the addition rate of the fed-batch medium. In other words, the rate of addition of the fed-up medium is limited by the rate at which the cells consume the substrate. Since the substrate consumption in the fermenter is determined by the specific growth rate and the cell weight of the cells, the high concentration of the cell increases the rate of consumption of the substrate, thereby increasing the speed of increase of the amount of the cell and thus improving the productivity of the recombinant protein.

However, in the case of the conventional fermented milk fermentation, the concentration of the cells is maintained at a low level for most of the incubation time, and high cell mass can be obtained only in the latter part of the fermentation. There was a problem of low. As a result, if the fermentation can maintain a high concentration of the cell mass continuously in the entire fermentation process, the productivity of the recombinant protein will be maximized.

To this end, Curless et al. Have developed a two-stage, repeatable fed-batch culture process that is a variant of fed-batch fermentation (Curless, C. et al., Biotechnology and Bioengineering, 38: 1082-1090 (1991). )), A certain volume of the culture medium of the cells cultured at high concentration in the growth stage of the fermenter is transferred to the fermenter of the second stage, that is, protein production stage, and the remaining culture is left in the fermenter of the first stage, the cell growth stage. Subsequently, in the fermenter in the protein production stage, the expression of the protein is induced, and the protein is mass-produced by the milking method required for protein production. During this time, the fermenter in the cell growth stage is again fed with the volume of the culture medium to be transferred to the second stage. Filled through By repeatedly performing such a process, a high concentration of cell mass was maintained throughout the fermentation process, thereby greatly improving the productivity of the re-controlled protein weaving.

In the present invention, by introducing such a two-step repeated feed culture process, to improve the productivity of recombinant G-CSF expressed under the L- arabinose inducible promoter. That is, in the first step for cell growth, an exponential feeding method is applied to keep the cell growth rate and dry cell weight constant at a high concentration. In the second step for production of recombinant protein, L-arabinose The induction of the recombinant protein production in a short time through the optimization of medium addition after induction, to improve the productivity of the recombinant protein.

The inventors of the present invention prior to the use of recombinant E. coli transformed with an expression vector containing an inducible promoter using L-arabinose operon, by performing an algebraic feeding method to control the specific growth rate of the cells before protein induction optimal growth After determining the rate, a process has been proposed to induce G-CSF expression with L-arabinose to obtain high cell mass and production of G-CSF. To increase the expression of granulocyte colony stimulating factor by fed-batch culture.

Based on this, in order to maximize G-CSF productivity, the present inventors made it possible to maximize production of G-CSF in a short time through optimization of medium addition after induction of expression of G-CSF, followed by two-step repetitive fed-batch culture. It was applied to the protein production stage in the process. That is, in the first stage of cell production, the cells are continuously grown in algebraic oil culture to maintain the specific growth rate and the specific growth rate by controlling the rate of addition of the feeding medium, and in the second stage of protein production, the first stage. Induction of protein expression by the L-arabinose of the cells delivered in the G-CSF by two-step repetitive fed-batch culture, which produces the protein in a constant feed-rate at a desired initial dilution rate in a medium containing glycerol as a carbon source. It was confirmed that the productivity was greatly improved, and the present invention was completed.

After all, an object of the present invention is to divide the fermentation step into a cell growth step of the first step and a protein production step of the second step, and to provide a method of improving the expression of recombinant G-CSF by repeating the two-step fermentation fermentation. It is.

FIG. 1 is a graph showing the results of algebraic fed-batch cultivation in which the cell growth stage, which is the first stage, of the two-stage repeat fed-batch cultivation is controlled to maintain a constant cell mass and a constant specific growth rate.

2 is a schematic diagram of an apparatus for carrying out a two-step, repeatable fed-rice fermentation.

3 is a graph showing the result of performing two-stage repetitive fermentation of 12 times.

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

Production of recombinant G-CSF using the L-arabinose inducible promoter by the two-step repeated fed fermentation process of the present invention, the amount of cells that are the fermentation process control variable of each cycle in the control of the two-stage repeated fed fermentation process And the amount of protein expression, etc. should be maintained at a constant high concentration, for this purpose, it is advantageous in terms of process control to maintain the constant cell concentration at a high concentration in the growth stage of the cells supplying the cells to the protein production stage.

That is, the most important thing in the cell growth stage of the two-stage repetitive fed-fermentation fermentation process is to continuously maintain a constant concentration of high concentration of cells growing at a constant specific growth rate. Thus, the present invention satisfies the above conditions using the following oil value method.

First, the exponential feeding fed-batch method is used to obtain the desired high cell mass at a constant specific growth rate after completion of the batch culture. The algebraic oil price method is carried out according to the following equation, and is carried out under the conditions of maintaining the specific growth rate of 0.13h ^-1 to 0.18h ^-1 determined at the desired rate. To increase the expression of granulocyte colony stimulating factor by fed-batch culture. The equation used in the algebraic fed-rate cultivation section is given by equation (1):

Wherein F ₀ is the addition rate of the fed-up medium (unit L / h); X ₀ is the initial cell mass (unit g / L); V ₀ is the initial culture volume (unit L); μ is the specific growth rate of the cells (unit h ⁻¹ ); Y _{X / S} is the yield of cells; S _fo is the glucose concentration in the fed medium; And t represents the logarithmic feed rate incubation time (unit h).

Second, after obtaining a high concentration of cell mass under a constant specific growth rate through the algebraic oil price method, the following oil price method is used to maintain the high concentration and specific growth rate.

① First, the growth of cells over time in the fed-batch culture section can be expressed by the following equation (2):

Wherein t is time (unit h); X is the cell mass (unit g / L); V is the fermented volume (unit L); And μ indicates the specific growth rate of the cells (unit h ⁻¹ ).

If the cells grow at a constant specific growth rate (μ), equation (2) can be represented by the following equation (3):

Wherein X ₀ is the initial cell mass (unit g / L); And V ₀ represents the initial fermentation volume (unit L).

At this time, if the concentration of the cells is kept constant, X = X ₀ , and equation (3) becomes the following equation (4), which is differentiated to obtain the final equation (5).

In the above formula, F ₁ represents the addition rate (unit L / h) of the fed-batch medium for maintaining a constant cell mass and a constant specific growth rate.

② In this case, the concentration of the limiting substrate in the fed-batch medium can be derived from the following equations. If the growth of the cells is sustained and there is no accumulation of other by-products, the yield of the cells from the substrate is as follows:

Where

Y _{X / S} is the yield;

r _X is the growth rate of the cells (unit g / L / h); And,

r _S represents the consumption rate of the substrate (unit g / L / h).

Therefore, Equation (2) may be expressed as Equation (7) below.

At this time, since the cell mass is constant, Equation (7) may be represented by the following Equations (8) and (9).

In addition, assuming that the limiting substrate of the fed-batch medium is used immediately without accumulation in the fermenter, the rate of consumption of the substrate can be expressed by the following equation (10):

Where

S _f1 represents the substrate concentration (unit g / L) of the fed-batch medium to maintain a constant cell mass and a constant specific growth rate.

Substituting Eq. (9) and Eq. (10) here into Eq. (6), the concentration of the limiting substrate in the fed-batch medium to maintain a constant cell mass and a constant specific growth rate is represented by the following equation (11):

Using formula (5), logarithmic fed-batch culture is carried out to maintain constant cell mass and constant specific growth rate. At this time, the cell mass (X) is preferably maintained at 45 g / L to 60 g / L, most preferably 51 g / L; Specific growth rate is preferably maintained to 0.13h ^-1 to 0.18h ^-1, and most preferably from 0.15h ^-1.

Using equations (1) and (5) above, it is confirmed that the cell mass, specific growth rate and stability of plasmid are kept constant through several cycles in the growth stage of the two-stage repetitive oil-feed culture. do. In addition, L-arabinose is added to induce the expression of the protein after six cycles of fed-batch culture in the growth stage, and confirms whether the expression of the protein is properly performed. Perform verification.

As described above in the cell growth step of the first step, while maintaining a constant high concentration of cells grown at a constant non-proliferation rate, while transferring a certain volume in the culture medium of these cells to the fermenter of the protein production step of the second step , which induces the expression of the protein and then the protein produced oil medium (culture medium of glycerol as a carbon source), the initial dilution rate 0.050h ^-1 to 0.120h ^-1, and most preferably from a recombinant by a constant-speed-Batch culture 0.085h ^-1 in for Mass production of G-CSF protein.

The volumetric productivity per unit volume of this two-stage fed fed fermentation is calculated by the following equation (12) when the fed fermentation is carried out by repeating n cycles:

Where

Pr _vol is the productivity per unit volume (unit g / L / h);

P _fzi is the concentration of the final protein produced in the protein production stage of the i cycle, in g / L;

V _fzi is the final fermentation volume (unit L) of the protein production stage of the i th cycle;

V _fli is the final fermentation volume (unit L) of the cell growth stage of cycle i;

t _d is the dead time (unit h);

t _b is the batch fermentation time (unit h);

t _g is the logarithmic feed rate of incubation time (unit h) to increase cell mass; And

t _fi represents the incubation time (unit h) of the i-th cycle.

In addition, as in the present invention, the final fermentation volumes (V _f1 , V _f2 ), the incubation time (t _f ), and the final protein concentration (P _f2 ) of the two stages are constant without change in each week period, and this cycle is infinite When repeated, equation (12) can be represented by the following equation (13):

The productivity per unit volume in the case of 12 cycles using the two-stage repetitive fed-batch culture of the present invention is based on the conventional oxygen-added Do-stat fed-batch culture method (refer to the same application, "Oxygenated DO-stat fed-batch formula Method for improving the expression of granulocyte colony stimulating factor by cultivation "and constant rate fed cultivation method after algebraic fed-batch cultivation (see" Improving the expression of granulocyte colony stimulating factor by the fed-batch culture controlling the specific growth rate of bacteria " It can be seen that compared to the productivity per unit volume when using ")"), more than two times and about 60% or more, respectively.

Hereinafter, the present invention will be described in detail through examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention by these examples is not limited.

Example 1 Optimization of Feeding Medium Addition at Growth Stage in Two-Step Repetitive Feeding Cultivation Process

In order to maintain constant cell mass and constant specific growth rate in algebraic feeding at the stage of cell growth, recombinant E. coli MC1061: pWAGF (KFCC-10961), which is a G-CSF expressing strain stored in 25% glycerol in a freezer (-70 ° C), is first used. (Reference: Korean Patent Application No. 97-15210) was incubated for 15 hours at 150 rpm in a 30 ℃ shaking incubator. This was aseptically inoculated as a fermenter BiofloIII (New Brunswick Sci. Co. Ltd., USA) of the initial culture volume 1L to a final concentration of 5% in the batch fermentation medium consisting of the composition of Table 1 below. Then, the batch was incubated at the initial operating conditions of neutral pH, temperature 37 ℃, stirring speed 600rpm and aeration amount 1.0vvm. At this time, the pH in the incubator was kept neutral with 15% (v / v) phosphoric acid as the acid, 10% (v / v) ammonium hydroxide as the base.

While culturing the cells in batch form in the initial culture medium, when all the nutrient components are depleted and the amount of dissolved oxygen rapidly increases, the logarithm of fed oil medium of Table 1 was added according to the formula (1). At this time, the specific growth rate (μ) is 0.15h ^-1 , the initial cell weight determined at the optimum rate in the "method of improving the expression of granulocyte colony stimulating factor by the fed-batch culture controlling the specific growth rate of cells" (X ₀ ) is 25g / L, initial culture volume (Vo) is 1L, glucose concentration (S _fo ) in fed medium is 400g / L, the yield of bacteria (Y _{X / S} ) 0.52 using algebraic fed-batch culture Was carried out.

TABLE 1

After 6 hours of algebraic oil value cultivation, when the cell mass reaches 51 g / L based on dry cell weight, 0.3 L of culture medium is harvested, and then, using the formula (5) in the remaining culture medium in the fermenter, constant cell mass and constant specific growth rate are maintained. Algebraic feeding medium (see Table 2) was added. In equation (5), 0.9 L of initial fermentation volume (V ₁ ) and 0.15 h ⁻¹ of specific growth (μ) were used. When the fermentation volume reaches 2.21 L, which is the maximum fermentation volume of the 2.5 L fermentation tank, every 6 hours, the culture medium is harvested by 1.3 L and the remaining culture medium 0.9 L is added with an algebraic feeding medium to maintain constant cell mass and constant specific growth rate. It was. In the above, the harvested culture solution was used as a sample for measuring the cell mass, glucose concentration, acetic acid concentration, plasmid stability.

Finally, after repeating the above cycle six times, L-arabinose is added to the remaining cultures to induce the expression of G-CSF, and using the DO-stat fed method for 12 hours to produce the protein of Table 3 below. Feeding medium was added to confirm the expression level of G-CSF.

TABLE 2

TABLE 3

In the above, the addition of the medium using advanced fermentation software (part No. M1182-0050, New Brunswick Sci. Co. Ltd., USA) on an IBM486DX2 computer connected to the fermenter via RS422 interface based on the induction formula used in the present invention. By adjusting. The cell weight was determined by absorbance measurement at 600 nm wavelength using a spectrophotometer (Ultrospec II, LKB, USA) and dry cell weight. The dry cell weight was obtained by centrifuging the sample to obtain only cells and washed with PBS (phosphate buffered saline). After repeating the centrifugation process twice, the obtained precipitate was suspended in distilled water and measured using a moisture analyzer (Mosture analyzer, Sartorius, MA40, Germany). The expression level of G-CSF was first measured by SDS-PAGE with 5 purified G-CSF standard substances of known concentration, and then stained, searched by a laser scanner, and analyzed by an image analyzer. The peak area of the stained part was determined by comparing it with a quantitative curve based on the peak area of the standard material. The stability of the plasmid was diluted 10 times in succession, smeared on the LB plate and incubated to form a single colony. It was determined as the degree of formation of a single colony showing ampicillin resistance. The adjustment of the dissolved oxygen in the fermenter was controlled by adjusting the stirring rate of the fermenter and the oxygen partial pressure ratio of the gas mixer. That is, every 30 seconds, dissolved oxygen collected through the IBM486DX2 computer connected to the fermentor via the RS422 interface was analyzed by advanced fermentation software (part No. M1182-0050, New Brunswick Sci. Co. Ltd., USA). Compared to 20%, which is not affected by the lack of oxygen, the amount of dissolved oxygen below this concentration increased the stirring speed of the fermenter. When the stirring speed reaches the maximum value of 970 rpm, the oxygen partial pressure in the aeration can be increased by using a gas mixer (New Brunswick Sci. Co. Ltd., USA) to maintain the dissolved oxygen amount in the fermenter at 20% or more. there was.

Fig. 1 shows the result of the algebraic oil price method in which the constant cell mass and the constant growth rate were maintained in the first stage of cell growth.

As shown in FIG. 1, the cell mass was kept constant for 6 cycles, the plasmid stability was maintained at a high level, and even after G-CSF expression was induced after 6 cycles, the expression of G-CSF was high. It was found to happen. Therefore, in the two-stage repetitive fed-batch cultivation, when the fed-batch cultivation is carried out according to the formula (5) using a fed-batch medium having the concentration of glucose calculated using Eq. It can be seen that it is maintained.

[Example 2: Optimization of Feeding Medium Addition in Protein Production Step in Two-Step Repeat Feeding Culture]

Since the method of Example 1 can stably control the cell growth stage of the two-step repetitive fed-batch fermentation process, in this example, it was intended to optimize the addition of fed-batch medium in the protein production step.

As can be seen in Example 1, in the 2.5L fermenter used in the present invention, when the cell mass of 51 g / L is maintained at a specific growth rate of 0.15h ^−1, the fermentation volume is 2.21, which is the maximum acceptable volume of the fermenter. Since it reaches L, in order to obtain the maximum protein yield at the protein production stage, a fed-batch culture strategy was needed to obtain the maximum protein yield 6 hours after the induction of protein expression.

After inducing the expression of the protein, constant-cost fed-culture to improve the expression of the protein. For this purpose, the batch fermentation was completed and then cultured until the cell mass reached 51 g / L by adding a fed medium for 6 hours while maintaining a specific growth rate of 0.15 h ⁻¹ using an algebraic feeding method. After the L-arabinose was added to induce the expression of G-CSF, constant-feed fed culture was carried out for 6 hours using the fed-value medium of Table 3 above. When glucose was used as the carbon source and glycerol was used, the optimum oil feed rate was determined by changing the rate of addition of the feed medium on the basis of the initial dilution rate (F / V ₀ ), and the results are shown in Table 4 below. Indicated.

TABLE 4

As shown in Table 4, when glucose is added as a carbon source as the initial dilution rate is increased, excess glucose is initially added to accumulate by-products such as acetic acid, and thus 0.085h ^-1 based on the initial dilution rate. In the case of adding the fed medium, the growth of the cells was severely inhibited and the expression of G-CSF was lowered compared to the amount of the cells. However, in the case of using glycerol as a carbon source, by-product accumulation, such as acetic acid, was not observed even when the fed medium was added at a high rate. As a result, the expression level of G-CSF was also expressed at a high level. . The optimum initial dilution rate was 0.085h ^{-1 for} the addition of a glycerol-based feedstock medium, and the dry cell mass and G-CSF expression levels after 7 hours were 73.4g / L and 9.02g / L, respectively. .

Example 3 2-Step Repetitive Fermented Oil Price

Using the oily medium having the composition shown in Table 2 above, the first stage of cell growth was controlled by an algebraic feeding method for maintaining a constant specific growth rate of 0.15 h ⁻¹ and a constant cell weight of 51 g / L (see Example 1). In Example 2, the protein production step of the second step by the constant dilution rate of the initial dilution rate of 0.085h ^-1 using the oil medium having the composition shown in Table 3 using the glycerol as a carbon source determined by the optimal method of induction after protein expression induction To control the two-stage, repeated feed fermentation was carried out. A simple schematic diagram of a two-step, repeatable fed fermentation method is shown in FIG.

In two fermenters, the first stage of cell growth and the second stage of protein production, cells were cultured in batch form in the batch fermentation medium of Table 1, each with an initial fermentation volume of 1 L. Since the amount of dissolved oxygen is rapidly depleted and the amount of dissolved oxygen is rapidly increased, the algebraic oil medium shown in Table 1 has been fed using Equation (1). At this time, the specific growth rate (μ) is 0.15h ^-1 , the initial cell mass (X ₀ ) is 25g / L, the initial culture volume (Vo) is 1L, the glucose concentration (S _fo ) in the fed medium is 400g / L, Yield (Y _{X / S} ) was algebraic fed-batch culture using 0.52 each.

After 6 hours of algebraic oil cultivation, the fermentation volume of 1.2L and the total cell weight were 51g / L based on the dry cell weight, 0.3L of the culture solution was transferred from the fermenter of the first stage to the fermenter of the second stage, followed by the first stage. In the fermenter, the algebraic feeding medium for maintaining a constant cell mass and a constant specific growth rate was fed using the formula (5) according to the method of Example 1. At this time, in the fermenter of the second step, L-arabinose is added to 1.5L culture medium containing 0.3L delivered in the first step to induce protein expression, and then the initial stage of 0.085h- ¹ according to the method of Example 2 Constant fed-batch culture was performed at the dilution rate. When the fermentation volume of the first stage reaches 2.21L, which is the maximum fermentation volume of the 2.5L fermentation tank, every 6 hours, the second stage culture medium (2.0-2.2L) is collected and 1.3L of the culture medium of the first stage is removed. It was delivered in two stages. After that, the algebraic feeding medium was added to 0.9L of the culture solution remaining in the fermenter of the first stage to maintain a constant cell mass and a constant specific growth rate.In the fermenter of the second stage, L was added to 1.3L of the culture medium delivered in the first stage. After inducing protein expression by adding arabinose, constant flow fed culture was performed at an initial dilution rate of 0.085 h ⁻¹ according to the method of Example 2.

The above cycle was repeated 12 times. At this time, samples were taken in the first and second stages at 6 hour intervals, and the cell mass, glucose concentration, acetic acid concentration, plasmid stability, and G-CSF expression levels were measured. . In the above, all pumps were designed to run at 6 hour intervals using a timer (ChronTrol, model CD-4, Lindburg Enterprise Inc., USA).

The results of the two-stage repetitive oil fermentation are shown in FIG. As shown in FIG. 3, it was found that, when 12 repeated oil fermentations were performed, cell mass, plasmid stability, and G-CSF expression levels were maintained at high levels in each of the two fermenters.

The result of comparing the productivity of the two-step repetitive fed-value fermentation method with the conventional fed-value fermentation method is shown in Table 5 below.

Productivity per unit volume by two-step repetitive fed-value fermentation of the present invention was calculated by equations (12) and (13), and the productivity per unit volume by conventional fed-value fermentation was calculated by the following equation:

Where

Pr _vol is the productivity per unit volume (unit g / L / h);

P _f is the concentration of the final protein produced (unit g / L);

t _d is the dead time (unit h);

t _b is the batch fermentation time (unit h); And,

t _f represents the fed-batch incubation time (unit h).

At this time, the fermentation preparation time, the dead time (t _d ), including the preparation and sterilization of the medium, the seed culture time, etc. were excluded from the calculation.

TABLE 5

a: the method disclosed in Korean Patent Application No. 97-15209.

b: The method disclosed in the same application filed "Method of Enhancing the Expression of Granulocyte Colony Stimulator by Oxygenated DO-stat Fed-Batch Culture".

c: The method disclosed in the same application filed "Method of Enhancing the Expression of Granulocyte Colony Stimulator by Feeding Cultivation Controlling the Specific Growth Rate of Cells".

d: Fermentation preparation time was excluded in the calculation of productivity.

e: In the case of the two-step repetitive feeding method, the incubation time after protein induction means the expression time per cycle.

As shown in Table 5, the productivity per unit volume in the case of performing 12 cycles using a two-stage repetitive fed-batch culture is 0.643 gL ^-1 h ^-1 , using the DO-stat fed-batch culture method with conventional oxygen More than double the productivity per unit volume of 0.281 gL ^-1 h ^-1 , and about 60% more than the 0.40 gL ^-1 h ^-1 productivity per unit volume when using a fixed-rate feed culture after algebraic oil cultivation Could know. In addition, this was found to be about 89% of the productivity of 0.728 gL ⁻¹ h ⁻¹ , which is an ideally infinitely repeated feed culture.

As described and demonstrated in detail above, in the method of the present invention, the cell mass, the stability of plasmid, and the expression level of G-CSF are all stably maintained at high levels in each fermenter in two stages, and the fermentation process is carried out repeatedly by n cycles. When performed, the productivity of G-CSF is also significantly improved compared to conventional methods.

Claims (5)

In the culture of recombinant E. coli transformed with the expression vector of granulocyte colony stimulating factor (G-CSF) using L-arabinose operon, the first stage of cell growth and the second step of protein production. Using two fermentors for stage fermentation, (i) in the first stage, algebraic fed ^- batch culture which controls the specific growth rate of cells from 0.13h- ¹ to 0.18h- ¹ using Equation (1) When the cell mass is 45 g / L to 60 g / L (dry cell weight basis), a part of the first stage fermentation broth is transferred to the second stage fermenter, and the remaining fermentation broth is 5) continue growing the cells in an algebraic fed-batch culture to keep the electrical specific growth rate and cell weight constant; (ii) In the second step, after the induction of protein expression by the L-arabinose of the cells delivered in the first step, constant rate fed culture at an initial dilution rate of 0.050h ^-1 to 0.120h ^-1 in a medium containing glycerol as a carbon source To improve the expression of recombinant granulocyte colony stimulator from electrorecombinant Escherichia coli by two-step repetitive fed fermentation, producing protein by:

Where F is the rate of addition of the feed medium (unit L / h); X ₀ is the initial cell mass (unit g / L); V ₀ is the initial culture volume (unit L); μ is the specific growth rate of the cells (unit h ⁻¹ ); Y _{X / S} is the yield of cells; S ₀ is the substrate concentration in the fed medium; t is the logarithmic feed time of incubation (unit t); V is the fermented volume (unit L); And, F ₁ represents the addition rate (unit L / h) of the fed-batch medium for maintaining a constant cell mass and a constant specific growth rate. The method of claim 1, wherein the recombinant microorganism is MC1061: pWAGF (KFCC-10961). The method according to claim 1, wherein in the first step of producing a cell, the cell weight is 51 g / L, and the specific growth rate is 0.15 h ⁻¹ . The method of claim 1, wherein the initial dilution rate in the second stage of protein production is 0.085 h ⁻¹ . The concentration of the limiting substrate in the fed-batch medium is controlled by the following formula (11) when carrying out algebraic fed-batch cultivation to maintain the specific growth rate and the cell mass constant using the formula (5). Characterized in that the method;

Where S _f1 is the substrate concentration (in g / L) in the fed medium; X is the cell mass (unit g / L); And, Y _{X / S} represents the yield of cells.

Images