KR100251283B1 - Method of improving expression of granulocyte colony stimulating factor by fed-batch culture that controls the specific growth rate of cells - Google Patents

Abstract

PURPOSE: Provided is a method for enhancing the expression of granulocyte colony stimulating factor G-CSF by constant feeding fed-batch that controls cell specific growth rate. Therefore, cell yield and expression of G-CSF are considerably enhanced and a plasmid stably is maintained in E. coli by latter stage of fermentation. CONSTITUTION: A method for enhancing expression of granulocyte colony stimulating factor G-CSF by constant feeding fed-batch is composed of the following steps of: (a) cultivating a recombinant E.coli transformed with G-CSF expression vector using L-arabinose operon of Salmonella spp. in high concentration; (b) inducing the expression of G-CSF using L- arabinose; (c) culturing by constant feeding fed-batch with adding feeding medium thereto.

Description

Method of improving expression of granulocyte colony stimulating factor by fed-batch culture that controls the specific growth rate of cells

The present invention relates to a method for enhancing the expression of granulocyte colony stimulating factor (G-CSF) by fed-batch culture that controls the specific growth rate of cells. More specifically, the present invention is a recombinant Escherichia coli transformed with a G-CSF expression vector using L-arabinose operon of Salmonella strain, high concentration as an algebraic feeding method (exponential feeding) to control the growth rate of the cells Incubated with L-arabinose to induce the expression of G-CSF, followed by the addition of a fed-batch medium for constant feeding fed-batch, thereby increasing the expression of recombinant G-CSF from recombinant E. coli. It is about.

In general, in the production of recombinant protein in recombinant E. coli, the growth of cells and the amount of expression per unit cell are greatly influenced by the characteristics of the promoter and the expressed protein.

In systems where the expression of proteins is regulated by powerful promoters, the rapid expression rate of the recombinant protein affects the balance of energy metabolism of the host cell and inhibits cell growth. And overexpression of the recombinant protein by a strong promoter in many cases lowers the stability of the plasmid. That is, a rapid decrease in plasmid occurs immediately after protein induction, accompanied by a decrease in recombinant protein expression rate.

The expression of the protein by a strong inducible promoter acts as a large burden on the host cell itself due to the above-mentioned causes, and the productivity of the recombinant protein depends on how appropriately mitigating such burden.

In a system using such a strong inducible promoter, a method for mass production of recombinant protein minimizes the inhibitory effects through optimization of protein expression induction timing and improves the productivity of a desired recombinant protein.

For example, in the expression system using the L-arabinose-inducible promoter used in the present invention, it was observed that the stability of the plasmid was reduced after inducing the expression of the protein. The induction period was determined as the time point at which the batch fermentation is complete and the fed-batch cultivation starts (see, “Improving the Expression of Granulocyte Colony Stimulator by Oxygenated DO-stat Fed-Batch Culture”, filed on the same day).

In general, in the period where batch fermentation is completed, since the cell mass is relatively small and the growth of cells is inhibited after protein induction, inducing protein expression in the latter part of the high-cost cultivation of high-value cell culture or mass production of protein It is advantageous in production.

However, according to the DO-stat fed-batch culture method proposed by the present inventors, when the induction period of the protein is delayed to the latter part of the fed-batch culture, and a high concentration of the cell mass is obtained to induce the expression of the protein, the batch fermentation in the protein expression or the cell mass is rather performed. Was found to be reduced than when inducing protein expression at the time of completion (see Korean Patent Application No. 97-15209).

It is predicted that the cause of this phenomenon is that the specific growth rate of the cells immediately before protein induction affects the expression of the recombinant protein (Curless, C. et al., Biotechnol. Prog., 6: 149-152). 1990). This predictability is based on the following: When cell growth is fast, the concentration of intracellular compositions that affect protein synthesis, such as active ribosomes, increases (Herbert, D., Symp. Soc. Gen, Microbiol). , 11: 391 (1961)), which means that fast growing cells can increase the rate of protein synthesis and maintain protein concentration and growth in the cells. For the same reason, if the growth rate of cells before protein induction is fast, recombinant proteins can be produced at high speed by using the composition in cells required for the synthesis of proteins present in high concentrations in cells.

When the cells are cultured using the DO-stat fed-batch culture method, the growth rate of the cells is remarkably slowed, and when the production of the protein is induced at this time, it can be considered that the above-mentioned protein expression or the decrease of the cell mass are caused. have.

The growth rate of the cells can be controlled by the addition rate of the medium during fermentation, but there is a problem that the addition rate of the medium cannot be artificially controlled in a feed-back process control system such as DO-stat.

Accordingly, the present inventors have developed a method of producing a large amount of G-CSF by maintaining the specific growth rate of the cells before induction of L-arabinose at an optimal rate for producing a large number of cells without accumulation of by-products. As a result of intensive research, the cells were cultured by an algebraic feeding method in which the cells before induction of L-arabinose were incubated at a constant specific growth rate, followed by inducing expression of G-CSF with L-arabinose, followed by the addition of a fed-rate medium. By fed-batch culture, it was confirmed that recombinant G-CSF was expressed at a high concentration from recombinant E. coli, thereby completing the present invention.

Finally, the object of the present invention relates to a method for improving the expression of recombinant G-CSF by algebraic fed-batch culture before L-arabinose induction and constant fed-batch culture after L-arabinose induction.

1 is a graph showing the fermentation pattern when the specific growth rate of the cells before the L-arabinose induction is 0.15h ^-1 and the DO-stat feeding method is performed after the induction.

FIG. 2 is a graph showing the dissolved oxygen, partial pressure of oxygen during aeration, and the consumption of oil medium when the specific growth rate of the cells before L-arabinose induction is 0.15h ⁻¹ and the DO-stat feeding method is performed after induction. to be.

FIG. 3 is a graph showing the fermentation improvement when constant-rate oil culture was performed at a rate of 0.15 h ⁻¹ before induction of L-arabinose and at a rate of 0.91 mL / min after induction.

The fourth turning L- arabinose dissolved oxygen, partial pressure and venting of the medium oil of the oxygen when the specific growth rate of the cells before induction hayeoteul subjected to the constant speed oil culture at a rate of 0.91mL / min and then to 0.15h ^-1, and the induction A graph showing the consumption of.

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

Since the recombinant microorganism transformed with the expression vector of G-CSF using L-arabinose operon was cultured in batch form in the initial culture medium, when the nutrient components were exhausted and the amount of dissolved oxygen rapidly increased, The medium is added and cultured by an algebraic feeding method. At this time, the relationship between the growth rate of the medium and the growth rate of the cells by the algebraic oil price method is the same as the final equation (7) calculated by the following series of formulas:

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

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), they can be represented by the following equation (2), which is substituted into equation (1):

Where

X ₀ is the initial cell mass (unit g / L); And,

V ₀ represents the initial fermentation volume (unit L).

In addition, the relationship between the rate of consumption of the substrate and the rate of oil consumption of the medium can be expressed by the following equation (4):

Where

S is the concentration of the substrate in g / L;

S ₀ is the substrate concentration (in g / L) in the fed medium; And,

F represents the addition rate (unit L / h) of the fed-batch medium.

Since S ≒ 0 in the quasi-steady state section during the incubation, Eq. (4) can be represented by the following equation (5):

In addition, the following formula (6) is established by the definition of yield:

Where

Y _{X / S} indicates yield of cell mass from the substrate.

Substituting equations (5) and (3) into equation (6) yields the following final equation (7):

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; And,

t denotes the logarithmic feed rate incubation time (unit h).

Choejongsik constant specific growth rate using 7, preferably 0.13h 0.18h ^-1 to ^-1, and most preferably for 5 to 6 hours incubation the cells were in the oil 0.15h ^-1, cells were not the accumulation of by-products After mass production, L-arabinose induces expression of recombinant protein G-CSF.

After L-arabinose induction, in order to mass-produce the recombinant protein G-CSF, a constant feed rate culture in which the medium is added at a constant rate, preferably 1.00 mL / min to 1.50 mL / min, most preferably 1.34 mL / min Perform constant feeding fed-batch.

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 is not limited by these examples.

Example 1 Control of Growth Rate of Cells Prior to L-Arabinose Induction

Recombinant Escherichia coli MC1061: pWAGF (KFCC-10961) (Korean Patent Application No. 97-15210), a G-CSF-expressing strain stored in 25% glycerol in a freezer (-70 ° C), was loaded at 150 rpm in a 30 ° C shaking incubator. Time incubation. 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, all of the nutrient components are depleted and the amount of dissolved oxygen rapidly increases, the algebraic fed oil medium of Table 1 was added.

After batch cultivation is completed, algebraic feeding method under various non-proliferation rates using final formula (7) (at this time, initial cell weight (X ₀ ) is 25g / L, glucose concentration (S ₀ ) in fed medium is 400g / L, Yield (Y _{X / S} ) is 0.52) and incubated cells for 5 to 6 hours, and then induces the expression of L-arabinose recombinant protein G-CSF, and thereafter, DO-stat milking method (Reference: Republic of Korea patent application 97-15209) is shown in Table 2 below.

As shown in Table 2, it can be seen that the optimum specific growth rate is 0.15h ^-1 , and when the addition rate of the medium is increased in order to maintain a higher specific growth rate, the accumulation of acetic acid in the algebraic oil incubation period is increased. It was observed that this caused a decrease in cell mass and expression yield of G-CSF. In the case of inducing protein expression at the optimal specific growth rate, the final cell weight and G-CSF expression amount were 6.62 g / L and 8.3 g / L, respectively, and the protein expression was induced immediately after the end of the batch fermentation period (control) Cell mass of 48.0 g / L and G-CSF expression of 6.8 g / L.

The fermentation pattern of the algebraic feeding method with an optimum specific growth rate of 0.15 h ⁻¹ is shown in FIG. 1. As shown in FIG. 1, it can be seen that the cell mass and medium consumption over time in the logarithmic oil price intervals are in good agreement with the results of computer simulations. It was found to be stable. These results were observed equally over all non-proliferative rates.

In the above, the cell weight was determined by absorbance measurement and dry cell weight at 600 nm wavelength using a spectrophotometer (Ultrospec II, LKB, USA), the dry cell weight is obtained by centrifuging the sample to obtain only the cells, which is PBS (phosphate buffered saline) After washing twice with centrifugation, the precipitate was suspended in distilled water and measured using a moisture 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 LB plates and incubated to form a single colony, which was then transferred to LB plates containing ampicillin at a concentration of 100 μg / ml and cultured. 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 fermenter via the RS422 interface was analyzed using advanced fermentation software (part No. M1182-0050), New Brunswick Sci. Co. Ltd., USA), and compared with 20%, the growth of the cells was not affected by the lack of oxygen, and increased the stirring speed of the fermenter if the amount of dissolved oxygen below this concentration decreases. When the stirring speed reached the maximum value of 970 rpm, by using a gas mixer (New Brunswick Sci. Co. Ltd. USA) to increase the oxygen partial pressure in the aeration, the amount of dissolved oxygen in the fermenter was maintained at 20% or more. . In addition, when the dissolved oxygen amount is excessively present at 70% or more after the stirring speed reaches the maximum value, the oxygen partial pressure ratio is reduced to prevent the consumption of excess oxygen.

Example 2 Optimization of Feeding Medium after L-Arabinose Induction

In high concentration cell culture and mass production of recombinant proteins, the addition of oxygen during fermentation is essential in terms of the prevention of by-product accumulation under anaerobic conditions and the inefficient use of substrates due to limited oxygen.

As described above in Example 1, when adjusting the addition of oxygen during fermentation, the oxygen partial pressure in the aeration can be used as an indicator for the growth of the cells during fermentation. In other words, increasing the partial pressure of oxygen during aeration means an increase in oxygen consumption, which means that the added medium is efficiently used for cell growth. On the other hand, when the partial pressure of oxygen in the aeration reaches a constant level and does not change or the partial pressure is continuously decreased, it means a decrease in growth rate or a decrease in cell mass due to the lack of medium or oversupply.

Based on this description, an attempt was made to optimize the method of adding fed medium after L-arabinose was added to induce the expression of G-CSF protein. Addition of medium prior to L-arabinose induction used an algebraic feeding method with a specific growth rate of 0.15 h ⁻¹ found to be optimal in Example 1.

FIG. 2 is a graph showing the dissolved oxygen, partial pressure of oxygen during aeration, and consumption of oil medium when the specific growth rate of cells before L-arabinose induction is 0.15h ^-1 and the DO-stat milking method is conducted after induction. . As shown in FIG. 2, when the DO-stat feeding method was performed after L-arabinose induction, the oxygen partial pressure in the aeration increased rapidly at the beginning and then maintained at a constant level of about 50% when reaching the latter part of the fermentation. there was. It was found that the growth of the cells and the expression of the protein occur rapidly immediately after the induction of L-arabinose, and in the latter part of the fermentation, the growth rate of the cells and the expression rate of the protein were inferior.

An experiment was conducted to see if this phenomenon could be improved by increasing the rate of addition of the medium. When the DO-stat oil-feeding method, which is a feedback control method for adding a medium using dissolved oxygen as an indicator, is not only easy to artificially control the rate of addition of the medium, but also the consumption of the medium as shown in FIG. Since the rate of growth increases constantly, the rate of addition of the medium is controlled at a constant rate, and the medium is added at the same rate as the rate of addition of the medium of DO-stat feeding method of FIG. 2, and compared with the result of DO-stat feeding method. It was.

FIG. 3 is a graph showing the fermentation pattern when the constant growth rate culture was performed at a rate of 0.15 h ⁻¹ before induction of L-arabinose at 0.15 h ⁻¹ and at 0.91 mL / min after induction. 4 shows the specific growth rate of the cells before induction of L-arabinose at 0.15h ^-1 , and the dissolved oxygen, partial pressure of oxygen during aeration, and oil medium at constant flow rate culture at a rate of 0.91 mL / min after induction. A graph showing the consumption of.

As shown in FIG. 3 and FIG. 4, the constant flow fed method at the rate of 0.91 mL / min showed that the consumption of oxygen and the medium were similar to those of the DO-stat fed method, but the final cell weight and The expression levels of G-CSF were increased to 65.4 g / L and 9.4 g / L, respectively.

Thus, the rate of addition of the medium was increased to 1.34 mL / min and 1.70 mL / min, respectively, to perform a constant flow method, and the results are shown in Table 3.

As shown in Table 3, the optimum oil price method after L-arabinose induction was found to be a constant injection of the medium at a rate of 1.34mL / min, when the medium is injected at a rate of 1.70mL / min of acetic acid Excessive accumulation inhibited the growth of the cells and no protein was expressed.

When fermentation was carried out at a rate of 1.34 mL / min after L-arabinose induction, the final cell weight and the expression level of G-CSF were 79.8 g / L and 10.2 g / L, respectively. At this time, the partial pressure of oxygen in the aeration was 65%.

As described and demonstrated in detail above, the method of the present invention (constant-cost oil culture method after algebraic oil price culture) is compared with conventional culture methods, for example, DO-stat-fuel culture method and DO-stat-value culture method after algebraic oil value culture, Significantly improves the expression level of G-CSF, and maintains the plasmid in E. coli stably until the second half of fermentation.

Claims (5)

When culturing recombinant microorganisms transformed with the expression vector of granulocyte colony stimulating factor (G-CSF) using L-arabinose operon, the specific growth rate of the cells was 0.13h ^-1 using the following formula. To perform the algebraic feed feeding (exponential feeding) to adjust to 0.18h ^-1 , and then induce the production of L-arabinose granulocyte colony stimulating factor, and then adding the milk medium at 1.00mL / min to 1.50 mL / min Methods to improve expression of recombinant granulocyte colony stimulator from electrorecombinant E. coli by constant feeding fed-batch:

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; And, t denotes the logarithmic feed rate incubation time (unit h). The method of claim 1, wherein the recombinant microorganism is MC1061: pWAGF (KFCC-10961). The method of claim 1, wherein the specific growth rate is 0.15 h ⁻¹ . The method of claim 1, wherein the medium addition rate after L-arabinose induction is 1.34 mL / min. The method according to claim 1, wherein the DO-stat fed value is cultured instead of the constant fed fed value culture after L-arabinose induction.

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