KR19990042953A - Method to improve expression of granulocyte colony stimulating factor by oxygenated DO-stat fed-batch culture - Google Patents

Abstract

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 of Salmonella strains. A method of maximizing efficiency and further enhancing expression of recombinant G-CSF from recombinant E. coli by inducing expression of the protein with L-arabinose at the time of induction of optimal expression, that is, at the end of batch culture and at the beginning of fed-batch culture. It is about. According to the present invention, even in the addition of oxygen, DO-stat fed-value cultivation, which is an indicator of the amount of dissolved oxygen, was efficiently performed, and was excellent in terms of cell mass, expression level of G-CSF, and stability of plasma. In addition, glucose added as a carbon source during the fed-batch culture was maintained below the minimum concentration of expression inhibition, and expressed a similar level of G-CSF as when using glycerol as a carbon source.

Description

Method to improve 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 of Salmonella strains. Maximizing the efficiency and further inducing the expression of the protein with L- arabinose 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 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 such a system using a strong inducible promoter, even when the same expression vector and the same host are used, the expression time may be different according to the characteristics of the expressed protein, and the timing of expression may also be different in the influence of 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 of 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 recombined by DO-stat oil-value fermentation method for mass expression of recombinant G-CSF Microorganisms have been cultured at 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.

Therefore, 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, transformed 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.

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 a 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.

FIG. 7 is a graph showing the concentrations of G-CSF and residual glycerol and glucose in culture medium over time when oxygen was added and DO-stat fed 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. ) And 2 (A) and 2 (B).

Under this background, the present inventors divided the batch into fermentation batch and the DO-stat fed-batch cultivation section to examine 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 certain level as compared with the case where no oxygen is added, and thus the concentration of by-products during the fermentation is also relatively accumulated, and the ash fermentation time is also increased. Significantly 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 amount of cells in accordance with the fermentation time also increased by more than 50% in the case of the addition of oxygen compared to the absence 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, the L-arabinose operon is a gene related to metabolizing arabinose, an pentose sugar, and is strongly inhibited by glucose at high concentrations corresponding to the glucose concentration necessary for high concentration culture of microorganisms. To date, the most commonly used carbon source for high concentration cultivation of microorganisms is glucose or glycerol. The most widely used glucose is known as the best carbon source in terms of growth yield of Escherichia coli, but is present at a high concentration in the medium as described above. In this case, it exhibits a strong inhibition of arabinose operon, and also produces acetic acid as a by-product, resulting in lowered protein production yield. 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 as a by-product of cell metabolism than 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. The DO-stat oil price method has been proposed (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 fed-batch culture was performed 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 compared to when glycerol is used as the carbon source. It can be seen that it is maintained to a similar degree in terms of.

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 G-CSF Mass Production.

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 used at 150 rpm in a 30 ° C shaking incubator. Time incubation. This was aseptically inoculated in a fermenter BiofloIII (New Brunswick Sci. Co. Ltd. USA) of 1 L of initial culture volume so as to have a final concentration of 5% in 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 amount 1.0vvm. At this time, the pH in the incubator was maintained 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).

Composition of basal medium for fed-batch cultivation of the present invention

TABLE 1

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 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 (A) is a graph showing the fermentation pattern in the ash fermentation section when oxygen is not added. FIG.

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, where a maximum of 5.5 g / L 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 at a certain level or higher, the dissolved oxygen amount could be maintained at 20% or more, thus acetic acid concentration during fermentation was also increased. It can be seen that the accumulation is relatively small at the maximum 1.7g / L, and 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, ash fermentation is finished by oil when the culture is not gyuncheryang in at the beginning of FIG addition of oxygen is the position the gyuncheryang is more than 60, when added to oxygen than inde 48 to OD ₆₀₀ reference, the efficiency of carbon source also know the higher Could.

In the above, the cell mass was measured by aseptically distilling the samples during fermentation step by step 10 times, and then measuring the absorbance at 600 nm using a spectrophotometer (LKB, Ultrospec II, USA).

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

As shown in FIG. 2 (A), even in the DO-stat oil-fed cultivation section, when oxygen was not added, the section in which dissolved oxygen decreased to 0% was continuously observed. 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 was found that the consumption rate of the added medium 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 the OD ₆₀₀ , and it was found that the cell weight was increased by more than 50% from the 98 on the basis of the OD ₆₀₀ when the oxygen was not added.

As a result, the addition of oxygen during the cultivation was shown to increase the efficiency of the DO-stat fed-batch culture, and also showed an excellent effect on the high concentration culture of the cells.

Example 2: Induction of G-CSF Protein Expression When Oxygen Added During Culture

In order to apply the results of Example 1 to high-concentration expression of G-CSF protein, the optimization of protein expression timing was examined.

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. After the fermentation samples were taken at regular time intervals, the concentration of cells, the expression level, and the stability of the plasmid, respectively.

In the above, the cell mass was measured by aseptically distilling the samples during fermentation step by step 10 times, and then measuring the absorbance at 600 nm using a spectrophotometer (LKB, Ultrospec 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 an LB plate and incubated to form a single colony, and then transferred to an LB plate 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.

FIG. 4 is a graph showing the cell mass and the stability of plasmid according to G-CSF protein induction time according to the fermentation time. 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 beginning of fed-batch culture showed the most desirable results in terms of cell mass, G-CSF expression level, and plasmid stability, and DO-stat fed-batch culture when oxygen was not added. However, the absolute level of G-CSF expression is increased by about 50% compared to the case without adding oxygen, which increases the amount of cells and increases the expression of G-CSF. I mean.

Example 3: Induction of G-CSF Protein Expression with Oxygen Added and Glucose as Carbon Source

In the batch fermentation section with high carbon source, glycerol is used as a carbon source to reduce the inhibitory effect of arabinose operon and the accumulation of acetic acid. Was developed (see Korean Patent Application No. 97-15209).

As shown in FIG. 2, when oxygen is added, the dissolved oxygen content is different from that when oxygen is not added. Therefore, even in the process in which the consumption speed of the feeding medium is fast, and thus the supply of the feeding medium is fast, in order to find out whether or not glucose can be efficiently used as a carbon source of the feeding medium, oxygen can be obtained. 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 stability of the plasmid of the cell mass expressed when the DO-stat was fed with oxygen and glycerol or glucose was used as a carbon source, respectively. FIG. 7 is a graph showing the remaining glycerol and glucose in the cultured G-CSF and the culture medium. It is a graph showing the concentration over time.

6 and 7, when cultured in the same manner as above, it was possible to maintain the concentration of glucose in the culture medium to 0.5g / L or less, G-expressed by the fed-batch culture using glucose 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-value cultivation using glucose as a carbon source of the fed medium under the condition of adding oxygen, it was confirmed that the growth of cells and intracellular plasmids are stably maintained, and the high G-CSF.

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 culture was maintained below the minimum concentration of expression inhibition, and expressed a similar level of G-CSF as when using glycerol as a carbon source.

Claims (4)

Stimulation of recombinant granulocyte colony from electrorecombinant Escherichia coli by the addition of oxygen in DO-stat fed culture of transformed microorganism transformed with the expression vector of granulocyte colony stimulating factor (G-CSF) using L-arabinose operon How to improve the expression of the factor. The method of claim 1, wherein the recombinant microorganism is MC1061: pWAGF (KFCC-10961). The method according to claim 1, wherein the protein induction by L-arabinose is carried out after 8 hours after fermentation, which is the start time of the fed-batch culture. The method of claim 1, wherein the culturing is carried out in a medium to which glucose is added as a carbon source.