KR100260099B1 - Method for propagating granulocyte colony stimulating factor - Google Patents

Abstract

PURPOSE: A method for improving the expression of granulocyte colony stimulating factor(G-CSF) by using oxygen added DO-STAT fed-batch fermentation is provided, thereby the expression of G-CSF can be improved regardless of the oxygen concentration. CONSTITUTION: The improvement of the expression of granulocyte colony stimulating factor(G-CSF) is accomplished: by adding L-arabinose after 8 hours of fermentation, when the fed-batch fermentation starts, in order to induce the mass production of recombinant granulocyte colony stimulating factor; and by adding oxygen, during the fed-batch fermentation of recombinant microorganism MC1061:pWAGF(KFCC-10961) which is transformed by the expression vector of granulocyte colony stimulating factor containing L-arabinose operon in a medium containing glucose as a carbon source.

Description

Method of enhancing expression of granulocyte colony stimulating factor by oxygenated DO-STAT fed-batch culture

The present invention relates to a method for enhancing the expression of granulocyte colony stimulating factor (G-CSF) by oxygenated DO-stat fed-batch culture. More specifically, the present invention relates to DO-stat fed-batch culture of recombinant Escherichia coli transformed with G-CSF expression vector using L-arabinose operon on Salmonella strains. Maximizing efficiency and further inducing the expression of L-Arabinose protein at the time of induction of optimal expression, that is, at the end of batch culture and at the beginning of fed-batch culture, greatly increasing the expression of recombinant G-CSF from recombinant E. coli. It is about a method.

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 protein is controlled by a strong promoter, 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 plasmidation occurs immediately after protein induction, and accompanied by a decrease in recombinant protein expression rate.

The expression of protein weaving by a potent 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 this burden.

In such a system using a strong inducible promoter, even when the same host vector and the same expression vector is used, the timing of expression may be different according to the characteristics of the expressed protein, and the timing of expression is different in the severity of the effect on cell growth and expression. . In order to mass produce recombinant proteins in such a system, the inhibitory effects can be minimized through optimization of protein expression induction timing, and productivity of a desired recombinant protein can be improved.

On the other hand, as a method for improving the productivity of recombinant protein has been proposed a method of culturing the high concentration of the cells producing the protein itself, in this context, the present inventors in the context of the recombinant microorganisms by the DO-stat oil fermentation method for mass expression of G-CSF Has been incubated at a high concentration (see Korean Patent Application No. 97-15209). The DO-stat fed-batch culture method uses a rapid increase in dissolved oxygen as a measure of depletion of nutrients in fermenters.

However, in the mass culture of Escherichia coli, the amount of dissolved oxygen is absolutely insufficient in the batch fermentation section of DO-stat fed-batch culture and in the DO-stat fed-batch culture section. This lack of dissolved oxygen changes the growth conditions of the cells from aerobic conditions to anaerobic conditions, forming by-products such as acetic acid, etc., and the limiting substrate required for growth of cells is oxygen, which is an electron acceptor rather than a carbon source. Due to this change, the added nutrients are not utilized efficiently, resulting in the growth of the cells depending on the oxygen concentration.

Accordingly, the present inventors have intensively researched to develop a method for mass production of recombinant G-CSF expressed under a strong inducible promoter by L-arabinose, and thus, transfected with a G-CSF expression vector using L-arabinose operon. In the DO-stat fed-rate culture of recombinant E. coli, oxygen is mixed and ventilated at a predetermined ratio to solve the above-mentioned undesirable phenomena due to the lack of oxygen, thereby greatly increasing the expression of recombinant G-CSF, and further, inducing the expression of protein. It was confirmed that the productivity of the recombinant protein can be improved through optimization, and the present invention has been completed.

As a result, an object of the present invention is to culture recombinant E. coli by DO-stat fed-batch culture with oxygen, and then to express recombinant G-CSF by adding L-arabinose at the start of fed-batch culture, which is the optimal expression induction time. It is to provide a way to improve.

Figure 1 (a) is a graph showing the fermentation pattern in the batch fermentation section when oxygen is not added.

Figure 1 (b) is a graph showing the fermentation pattern in the batch fermentation section when oxygen is added.

FIG. 2 (a) is a graph showing the change of dissolved oxygen between 12 and 13 hours in the fed-batch culture section when oxygen is not added.

FIG. 2 (b) is a graph showing the change of dissolved oxygen between 12 and 13 hours in the fed-batch culture section when oxygen is added.

3 is a graph showing the change in cell mass with time when oxygen is not added.

FIG. 4 is a graph showing the cell mass and the stability of plasmid according to the G-CSF protein induction time in the DO-stat high fed-batch culture with oxygen.

FIG. 5 is a graph showing the expression of G-CSF protein according to the fermentation time according to the G-CSF protein induction time in oxygen-containing DO-stat high fed-batch culture.

FIG. 6 is a graph showing the cell mass and the stability of plasmids when DO-stat oil value culture was carried out by adding oxygen and glycerol or glucose as a carbon source, respectively.

7 is a graph showing the concentration of G-CSF and residual glycerol and glucose in the culture medium over time when oxygen is added and the DO-stat is incubated with glucose or glycerol as a carbon source.

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

As described above, in the mass culture of Escherichia coli, the amount of dissolved oxygen is absolutely insufficient in the batch fermentation section of DO-stat fed-batch culture and in the DO-stat fed-batch culture section. This is clearly demonstrated in Figure b) and Figures 2 (a) and 2 (b).

Under these backgrounds, the present inventors divided the batch into fermentation batch and DO-stat fed-batch cultivation section to test whether the addition of oxygen during the cultivation increases the efficiency of the DO-stat fed-batch cultivation, and experimented according to the presence or absence of oxygen addition. As a result, when oxygen is added in the batch fermentation section, the amount of dissolved oxygen in the culture is maintained at a predetermined level or more, compared to the case where no oxygen is added, so that the concentration of by-products during the fermentation is relatively deposited, and the ash fermentation time is also increased. Greatly shortened. In addition, the cell mass at the end of the batch fermentation and the start of the cultivation of oil was also significantly increased compared to the case where no oxygen was added, and the efficiency of the carbon source was also increased. Oxygen deficiency was not observed in the DO-stat fed-rate cultivation section, and the rate of consumption of the added medium was also faster than in the case of no addition of oxygen. In addition, the cell weight according to the fermentation time was increased by 50% or more in the case of adding oxygen compared to the case without the addition of oxygen.

Therefore, it was found that the addition of oxygen during the cultivation increased the efficiency of the DO-stat fed-batch cultivation and had an excellent effect on the high concentration cultivation of the cells.

Subsequently, in the use of DO-stat fed-batch culture method in which oxygen was added during fermentation, as a result of determining the optimal induction time, when production of recombinant G-CSF was induced at the time of batch culture and fed-batch start, Most preferred in terms of cell mass, expression level of G-CSF and stability of plasmid. In particular, the amount of G-CSF expression was increased by about 50% compared to the case of not adding oxygen, indicating that the addition of oxygen has a significant effect on the expression of G-CSF with the increase of the cell mass.

Meanwhile, L-arabinose operon is a gene related to metabolizing arabinose, an pentose sugar, and is strongly inhibited by glucose at high concentrations corresponding to glucose concentrations at high concentrations of microorganisms. To date, the most commonly used carbon source for high concentration culture of microorganisms is glucose or glycerol. The most widely used glucose is known to be the best carbon source in terms of growth yield of E. coli, but is present in high concentration in the medium as described above. In this case, it shows a strong inhibition of arabinose operon, and also produces acetic acid as a by-product, resulting in lowering the yield of protein weave. For this reason, in the recombinant E. coli of the present invention transformed with a G-CSF expression vector using L-arabinose operon, glycerol was used as a carbon source in a batch fermentation section in which high concentrations of glucose were present. However, glycerol does not inhibit arabinose operon and has the advantage of producing less acetic acid, a byproduct of cellular metabolism, compared to glucose, but has a disadvantage in that the growth rate of cells is low and expensive.

DO-stat, the oil price culture method used in the present invention, is a method of cultivating oil value by recognizing the depletion of the carbon source in the fermenter as the amount of dissolved oxygen, which can prevent the accumulation of the carbon source, such as acetic acid produced There is an advantage to prevent the accumulation of by-products.

Therefore, the present inventors use glycerol as a carbon source in a batch fermentation section in which carbon sources are present at a high concentration, thereby reducing the inhibitory effect of arabinose operon and the accumulation of acetic acid, and using glucose as a carbon source which is inexpensive and has high utilization efficiency in a fed-batch culture section. Has proposed a DO-stat oil price method (see Korean Patent Application No. 97-15209)

In order to determine whether the above-mentioned advantages are also shown in the process of rapid consumption of the feeding medium and the supply of the medium (DO-stat oil feeding with oxygen addition during the culture), glucose as a carbon source of the feeding medium under the addition of oxygen DO-stat oil price culture was carried out using. As a result, even in the step of adding oxygen, the residual glucose concentration in the medium is kept below the minimum concentration of expression inhibition, and the cell growth, the plasmid stability, and the G-CSF expression amount are compared with those of glycerol as a carbon source. It can be seen that the aspect is maintained to a similar degree.

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

[Addition of Oxygen in DO-stat Fed-Batch Culture for Mass Production of G-CSF]

Recombinant Escherichia coli MC1061: pWAGF (KFCC-10961) (see Korean Patent Application No. 97-15210) of G-CSF expressing strain stored in 25% glycerol in a freezer (-70 ° C) at 15 rpm at 30 ° C shaking incubator Time incubation. This was aseptically inoculated into a fermenter BiofloIII (New Brunswick Sci. Co. Ltd., USA) of 1 L of initial culture volume to a concentration of final 5% of the initial 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 rate 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.

After culturing the cells in batch form in the initial culture medium, when all the nutrient components were depleted and the amount of dissolved oxygen rapidly increased, the fed medium of the following Table 1 was added and cultured by DO-stat feeding method (see: Korean Patent Application No. 97-15209). The oxygen addition in the aeration was automatically controlled using a gas mixer (New Brunswick Sci. Co. Ltd., USA).

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 with the 20% concentration which is not affected by the lack of oxygen, the amount of dissolved oxygen decreases below this concentration increases the stirring speed of the fermenter. 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. .

FIG. 1 (a) is a graph showing the fermentation pattern in the ash fermentation section when oxygen is not added, and FIG. 1 (b) is an oxygen addition.

As shown in FIG. 1 (a), in the absence of oxygen, a section was formed in which the amount of dissolved oxygen was reduced to 0% due to the rapid consumption of oxygen in the batch fermentation section. Accumulation of by-products such as acetic acid appeared, and the growth rate of the cells was relatively slow, so that the batch fermentation period was maintained for about 9 hours, and then the cultivation of the fed-batch was observed.

On the other hand, as shown in FIG. 1 (b), when oxygen was added to maintain the dissolved oxygen level above a certain level, the dissolved oxygen amount could be maintained at 20% or more, thus acetic acid concentration during fermentation was also maximized. It was found that the accumulation of relatively less as 1.7g / L, the time of the batch fermentation section is also reduced to more than 1 hour 30 minutes to about 7 hours 20 minutes. In addition, when the batch fermentation is finished and the fed-batch culture starts, the cell mass is also 0D when oxygen is not added.₆₀₀ Compared to 48 as a standard, when the oxygen was added, the cell weight was 60 or more, indicating that the efficiency of the carbon source was increased.

In the above, the cell mass was determined by aseptically retaking the sample during fermentation and diluting it 10 times sequentially, and then determining the absorbance at 600 nm using a spectrophotometer (LKB U1trospec II, USA).

In addition, FIG. 2 (a) is a graph showing the change of dissolved oxygen between 12 and 13 hours in the fed-batch culture section when oxygen is not added and FIG. 2 (b) is the oxygen addition.

As shown in FIG. 2 (a), even when DO-stat is not added to the culture zone, a section in which dissolved oxygen drops to 0% was observed continuously. It was found that the rate of consumption was determined by insufficient oxygen, resulting in a slow rate of consumption.

On the other hand, as shown in Figure 2 (b), when the oxygen is added, such a lack of oxygen did not appear, it can be seen that the added consumption rate is also faster than without the addition of oxygen. This means higher growth of the cells when oxygen is added during the same fermentation time.

As a result, the change in cell mass according to the fermentation time is shown in FIG. As shown in FIG. 3, when the oxygen was added, the cell weight was 150 based on 0D ₆₀₀ , and it was found that the cell weight was increased by 50% or more than the 98 on the basis of 0D ₆₀₀ when the oxygen was not added.

As a result, the addition of oxygen during the cultivation increased the efficiency of the DO-stat fed-batch culture, and also showed an excellent effect in the high concentration culture of the cells.

Example 2

[Induction of G-CSF Protein Expression When Oxygen Added during Culture]

In order to adapt the results of Example 1 to the high concentration expression of G-CSF protein we examined the optimization of protein expression timing.

For this purpose, the concentration of final L-arabinose was 1% (w / w) at the logarithmic growth time during the DO-stat fed-batch cultivation, at the start of the fed-batch cultivation, and at about 4 and 8 hours after the start of the fed-batch cultivation. v) fed into the fermenter aseptically, and fed a value-induced culture to induce G-CSF expression, respectively. Thereafter, fermentation samples were taken at regular time intervals, and the concentration of cells, expression level, and stability of the plasmid were respectively confirmed.

In the above, the cell mass was measured by aseptically taking samples during fermentation, diluting them 10 times sequentially, and then measuring the absorbance at 600 nm using a spectrophotometer (LKB, U1trospec II, USA).

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.

4 is a graph showing the cell mass and the stability of plasmid according to the G-CSF protein induction phase in accordance with fermentation time in the DO-stat high fed fed-batch culture with oxygen, and FIG. This is a graph.

As shown in FIG. 4 and FIG. 5, the expression level of G-CSF was maintained high when the expression was induced during the logarithmic growth period in the initial culture medium, but the stability of plasmid was rapidly decreased at the end of fermentation. On the contrary, when DO-stat induction of protein production was induced later in the culture, it was found that the expression was not properly performed.

From these results, it was found that the optimal protein induction time is the point at which the batch culture and the fed-batch culture start. When protein production was induced at this time, the protein expression was also maintained at a high concentration, and the stability of the plasmid was also maintained at a high level until the late fermentation. As described above, inducing G-CSF expression at the start of fed-batch culture showed the most desirable results in terms of cell mass, G-CSF expression level, and plasmid stability. In the same way, the absolute level of G-CSF expression was increased by about 50% compared to the case without adding oxygen, which increased the cell mass and contributed to the expression of G-CSF. Means that.

Example 3

[Induction of G-CSF protein expression when oxygen is added and glucose is a carbon source]

DO-stat fed-batch culture method using glycerol as a carbon source to reduce the inhibitory effect of arabinose operpon and accumulation of acetic acid in the batch section where carbon source is present at high concentration, and low-cost and high-efficiency glucose as a carbon source Was developed (see Korean Patent Application No. 97-15209).

As shown in FIG. 2, the addition of oxygen shows a different aspect of dissolved oxygen than does not add oxygen. Thus, even in a process in which the consumption rate of the fed-rate medium is fast, and thus the supply of the organic medium is rapidly progressed, in order to find out whether the glucose can be efficiently used as a carbon source of the fed-up medium to have the above-mentioned advantages, DO-stat fed-batch culture was performed using glucose or glycerol as a carbon source of the fed medium under the added condition.

FIG. 6 is a graph showing the cell mass and the stability of plasmid when DO-stat was fed with the addition of oxygen and glycerol or glucose as a carbon source, respectively. FIG. 7 is the concentration of remaining glycerol and glucose in the expressed G-CSF and the culture medium. Is a graph with time.

As shown in FIG. 6 and FIG. 7, when cultured in the same manner as described above, the concentration of glucose in the culture medium was maintained at 0.5 g / L or less, and G-expressed by fed-batch culture using glucose was expressed. The amount of -CSF was the same expression level as when using glycerol as a carbon source, and the growth of cells and stability of plasmid were not significantly different from those when using glycerol as a carbon source.

Therefore, DO-stat fed-batch cultivation using glucose as a carbon source of the fed medium under the condition of adding oxygen, stably maintained the growth of cells and the intracellular plasmid, and the expression level of G-CSF was also high. .

As described and demonstrated in detail above, according to the present invention, even in the addition of oxygen, DO-stat fed-batch cultivation, which is an indicator of the amount of dissolved oxygen, is efficiently performed, and in terms of cell mass, expression level of G-CSF, and stability of plasmid Excellent at In addition, glucose added as a carbon source during the fed-batch cultivation was kept below the minimum concentration of expression inhibition, and expressed a similar level of G-CSF as when using glycerol as a carbon source.

Claims (2)

When fed the DO-stat of the recombinant microorganism MC1061: pWAGF (KFCC-10961) transfected with the expression vector of granulocyte colony stimulating factor (G-CSF) using L-arabinose operon, the fed-batch culture was started. 8 hours after the fermentation, which is a time point at which the fermentation is performed, L-arabinose is added to induce mass production of recombinant granulocyte colony stimulating factor, and oxygen is added to improve expression of recombinant granulocyte colony stimulating factor from E. coli. The method of claim 1, wherein the culturing is carried out in a medium to which glucose is added as a carbon source.

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