KR100211288B1 - Do-stat cultivation method for the mass production of gsf - Google Patents

Abstract

The present invention relates to a DO-stat fed-batch culture method for mass production of granulocyte colony stimulating factor (G-CSF). More specifically, the present invention is a batch culture of recombinant E. coli containing an inducible G-CSF expression vector using L-arabinose operon of Salmonella strains in an initial culture medium, the amount of dissolved oxygen rapidly increases The present invention relates to a DO-stat fed-batch cultivation method for mass production of G-CSF, including the step of culturing in a fed medium and adding L-arabinose at the beginning of the fed culture. According to the DO-stat fed-batch fermentation method of the present invention, in the batch fermentation section in which carbon source is present at high concentration, glycerol is used as the carbon source to reduce the inhibitory effect of arabinose operon and the accumulation of acetic acid, and to measure the amount of dissolved oxygen. In the oil-cultivation section, the problem of inhibiting G-CSF expression inhibition was solved by maintaining the low-cost, high-efficiency glucose as the carbon source below the expression inhibition minimum concentration, and adding L-arabinose at the beginning of the oil-cultivation. Thus, the growth of cells, stability of plasmid and expression level of G-CSF are stably maintained at a high level, thereby mass-producing G-CSF at low production cost.

Description

DO-stat fed-batch culture method for mass production of granulocyte colony stimulating factor

The present invention relates to a dissolved oxygen (DO) -stat fed-batch culture method for mass production of granulocyte colony stimulating factor (G-CSF). More specifically, the present invention is to culture the recombinant Escherichia coli containing an inducible G-CSF expression vector using L-arabinose operon of Salmonella strain, in a batch culture in an initial culture medium containing a carbon source other than glucose , L- cultivation at the start of milk culture, which is the optimal expression induction period selected in consideration of cell growth, plasmid stability, and G-CSF expression level, while cultivating in fed medium from the rapid increase of dissolved oxygen. It relates to a DO-stat fed-batch culture method for mass production of G-CSF, including the process of adding aribinose.

In general, in the production of recombinant protein by recombinant E. coli, the growth of cells and the amount of expression per unit cell are affected by the properties of the promoter and the expression protein. In systems where the expression of proteins is controlled by a strong promoter, the rapid expression of recombinant proteins may affect the balance of energy metabolism of the host cells and inhibit cell growth. In particular, the expression proteins directly affect host cells. In this case, cell growth is further inhibited.

In addition, in the production of recombinant proteins, overexpression of the protein by a strong promoter in many cases lowers the stability of the plasmid. That is, immediately after the induction of the protein, a sharp decrease in plasmid occurs, and it is reported to decrease with the expression rate of the recombinant protein (Park, SH et al., Biotechnol. And Bioeng., 36: 493). (1990)).

The expression of the protein by the strong inducible promoter causes a large burden on the host cell itself due to the above-mentioned causes. The minimization of the burden results in mass production of the recombinant protein. Even when the same expression vector and the same host cell are used in consideration of the conditions described above, the timing may vary depending on the characteristics of the expressed protein, and the timing of expression may vary depending on the effect of cell growth and expression. Can be.

In order to mass produce recombinant proteins in such a system, the inhibitory effects can be minimized through optimization of the fermentation process, and the productivity of the desired recombinant protein can be improved.

Meanwhile, human granulocyte colony stimulating factor (G-CSF) promotes the proliferation and differentiation of neutrophil progenitor cells to increase neutrophil release, and also activates mature neutrophils to activate phagocytosis, antibody-dependent cell killing, and super It is known to increase the formation of oxides (superoxide), the response to chemotactic factors (chemotactic factor). In addition, G-CSF has been developed as a drug to improve the neutrophil deficiency that is a side effect of chemotherapy, forming a large market worldwide.

In order to mass-produce such important G-CSF, the present inventors have already prepared an inducible G-CSF expression vector pGW2 using L-arabinose operon and introduced it into Escherichia coli, transforming E. coli MC1061: pWAGF (KFCC). -10961) (see Recombinant Microorganism Expressing Granulocyte Colony Stimulating Factor and Method for Producing Electron Recombinant Protein therefrom). However, batch culture of the electrical transformants did not produce satisfactory levels of presynthetic G-CSF.

Therefore, the present inventors have made intensive studies to improve the productivity of G-CSF through optimization of the fermentation process. As a result, the recombinant E. coli MC1061: pWAGF (KFCC-10961) was added to a new initial culture medium containing a carbon source other than glucose. When nutrients are depleted and the amount of dissolved oxygen rapidly increases, cultivating DO-stat in a new fed medium, and simultaneously adding L-arabinose to induce protein expression leads to cell growth, It was confirmed that the stability of the plasmid and the expression level of recombinant G-CSF remained stable at a high level, thereby completing the present invention.

After all, it is an object of the present invention to provide a DO-stat fed-batch culture for mass production of G-CSF.

1 is a graph showing the fermentation pattern of a typical DO-stat fed-batch culture.

FIG. 2 is a graph showing cell growth and plasmid stability according to fermentation time according to granulocyte colony stimulating factor (G-CSF) protein induction in DO-stat high fed-batch culture.

FIG. 3 is a graph showing the expression of G-CSF protein according to the fermentation time according to the G-CSF protein induction time in DO-stat high fed-batch culture.

FIG. 4 is a graph showing cell concentration and plasmid stability when DO-stat is fed with a glucose or glycerol as a carbon source according to fermentation time.

5 is a graph showing the concentrations of G-CSF and glucose remaining in the culture medium and the concentration of glycerol and glucose in the culture medium when DO-stat was fed with glucose or glycerol as a carbon source according to fermentation time.

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

Initial culture medium containing carbon source other than glucose for mass production of G-CSF from recombinant E. coli (KFCC-10961) containing inducible G-CSF expression vector using L-Aribinose operon of Salmonella strain Preferably, the recombinant Escherichia coli is cultured in a batch in a medium consisting of yeast extract 40g / L, carbon source 40g / L, Na ₂ HPO ₄ 10.22g / L, KH ₂ PO ₄ 2.31g / L and ampicillin 0.07g / L, DO-stat fed-batch cultivation with added oil medium (media consisting of yeast extract 292g / L, carbon source 400g / L, anhydrous magnesium sulfate 5.76g / L) from depletion of nutrients Use the bum. In this case, the carbon source of the initial medium is most preferably glycerol, and the carbon source of the fed medium is most preferably glucose.

DO-stat fed-batch cultivation uses dissolved oxygen as an indicator of depletion of nutrients in fermenter, and supplements nutrients when dissolved oxygen increases above a certain level, and then reduces dissolved oxygen with consumption of supplemented nutrients. If it increases again due to the depletion of the nutritional component, it means the oil price method to replenish the nutritional component again. In this way, E. coli, which can use the dissolved oxygen as an indicator of nutrient depletion, oxidizes the nutrient represented by the carbon source under aerobic conditions and decomposes it into a low-molecular substance, which is used as an electron acceptor in the corresponding action of the carbon source. This is because it uses. That is, the rate of oxygen consumption is proportional to the rate of consumption of nutrients in the medium, and depletion of nutrients causes a decrease in the rate of oxygen consumption, leading to a rapid increase in the amount of dissolved oxygen.

포환 이상일 때 유가배지를 주입하고, 50

이하로 떨어질 때 배지 주입을 중단하는 DO-stat 유가법을 실시한다.In the present invention, dissolved oxygen is measured by measuring the amount of dissolved oxygen in the culture medium.

When the shot is over, inject the oil medium, 50

DO-stat feeding is performed to stop media injection when it falls below.

In the DO-stat fed-batch culture of the recombinant Escherichia coli (KFCC-10961) described above, the timing of induction of protein expression in which cell growth, stability of plasmid and expression level of recombinant G-CSF can all be stably maintained Since it is important to determine, the L-arabinose was added to induce the expression after the logarithmic growth of batch fermentation, the start of DO-stat fed-batch culture and the completion of the fed-batch culture, and then the cell concentration, stability of plasmid and The expression level of recombinant G-CSF was measured. As a result, the optimal induction period was found to be the starting point for DO-stat oil price culture.

On the other hand, L-arabinose operon is a gene related to the target of arabinose, an octane sugar, and when glucose is present at a certain concentration or more, it receives strong suppression (catabolite repression). By the way, high concentration culture of microorganisms necessarily require a large amount of carbon source, and to date, the most commonly used carbon source for high concentration culture of microorganisms is glucose or glycerol. Glucose, the most widely used carbon source, is known to be the best carbon source in terms of growth rate and yield of E. coli, but when present in a high concentration in the medium, it exhibits a strong inhibition of arabinose operon as described above, and also a by-product. As a result, a large amount of acetic acid is produced, resulting in a decrease in the yield of protein. In view of this point, in the case of the present invention using arabinose operon, glycerol is used as a carbon source in a batch fermentation section in which high concentration of glucose is present. However, glycerol does not inhibit arabinose operon and produces less acetic acid, a byproduct of cell metabolism, compared to glucose, but has a disadvantage in that the growth rate of cells and the price are high.

DO-stat, the oil value culture method used in the present invention, recognizes the depletion of the carbon source in the fermentation tank as an increased amount of dissolved acid pine, and carries out the oil value culture, which prevents the accumulation of carbon sources and overcomes the above-described expression inhibition of glucose. And at the same time prevent the accumulation of by-products such as acetic acid caused by the accumulation of carbon sources.

Under this background, in the present invention, glycerol is used as a carbon source in a batch fermentation section in which carbon sources are present in high concentrations, thereby reducing the inhibitory effect of arabinose operon and accumulation of acetic acid, and inexpensive and excellent in utilization efficiency as a carbon source. Glucose was maintained at or below the expression suppression minimum concentration, preferably 0.5 g / L or less, thereby producing a large amount of G-CSF without expression inhibition compared to when glycerol was used as the carbon source.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only to specifically describe 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

[DO-stat Fed-Batch Culture for G-CSF Production]

)에서 25

글리세롤에 보관된 G-CSF 발현균주인 재조합 대장균 MC1061:pWAGF(KFCC-10961)(참조: 동일자 출원하는 『과립구 콜로니 자극인자를 발현하는 재조합 미생물 및 그로부터 전기 재조합 단백질의 제조방법』)를 30

진탄 배양기에서 150 rpm으로 15시간 배양한 다음, 하기 표1의 조성으로 이루어진 초기배지에 최종 5

(v/v)의 농도가 되도록, 초기 배양부피 1L인 발효조 Biofio Ⅲ(New Brunseick Sci. Co. Ltd., USA)에 무균적으로 접종하였다.In order to cultivate the G-CSF expressing strain in a DO-stat fed-batch, first, freezer (-70)

25 from

Recombinant Escherichia coli MC1061: pWAGF (KFCC-10961) (cf., `` Recombinant microorganism expressing granulocyte colony stimulating factor and method for producing an electric recombinant protein therefrom '', which is a G-CSF expressing strain stored in glycerol)

After incubation at 150 rpm in a shaker incubator for 15 hours, the final 5

Inoculated aseptically into the fermenter Biofio III (New Brunseick Sci. Co. Ltd., USA), which is 1 L of the initial culture volume, to have a concentration of (v / v).

, 교반속도 600rpm, 통기량 1.0vvm으로 하였고, 이후 용존 산소량을 최소 포화 대비 10

이상으로 유지시켜 주기 위하여 초기 배지성분이 고갈될 때까지 지속적으로 교반 속도를 증가시켰다. 이때, 배양기 내의 pH는 15

(v/v) 인산과 10

(v/v) 수산화 암모늄을 사용하여 중성으로 유지시켰다.Initial operating conditions are neutral pH, temperature 37

, Stirring speed 600rpm, aeration rate 1.0vvm and then dissolved oxygen amount to 10

In order to maintain the above, the stirring speed was continuously increased until the initial medium component was depleted. At this time, the pH in the incubator is 15

(v / v) phosphoric acid and 10

(v / v) Ammonium hydroxide was used to keep neutral.

포화 이상일 때 유가 배지를 주입하고, 50

이하로 떨어질 때 배지 주입을 중단하는 DO-stat 유가법을 자동으로 조절하여 배양이 끝날 때까지 반복하였다. 제1도는 DO-stat 유가법을 이용하는 경우의 전형적인 발효양상을 나타낸다.After culturing the recombinant E. coli in a batch culture in the initial culture medium, the nutrient components are all depleted and the dissolved oxygen is rapidly increased, and the fed medium of the following Table 1 is added, and cultured by DO-stat fed-batch. That is, the dissolved oxygen in the culture medium is 50

Infuse the feeding medium when above saturation, 50

DO-stat feeding method to stop the medium injection when falling below was automatically adjusted until the end of the culture. Figure 1 shows the typical fermentation pattern when using the DO-stat feeding method.

Example 2

[Induction of G-CSF Protein Expression]

(w/v) 되도록 무균적으로, 발효조에 각각 첨가하여 G-CSF 발현을 유도하였다. 이후 일정한 시간 간격으로 발효 시료를 채취하여, 세포 농도, 플라즈미드의 안정성 및 G-CSF 발현량을 각각 조사하였다.To find out whether expression induction at DO-stat fed-batch culture maximizes cell concentration, plasmid stability, and G-CSF expression, logarithmic growth point and fed-batch culture during DO-stat fed-batch culture At the beginning of the run, about 4 hours and 8 hours after the start of the oil price, the final concentration of L-arabinose is 1

(w / v) Aseptically, each was added to the fermenter to induce G-CSF expression. Thereafter, fermentation samples were taken at regular time intervals, and cell concentration, plasmid stability, and G-CSF expression levels were examined.

In the above, the concentration of the cells was determined by sequentially diluting the collected sample 10 times, and then measuring the absorbance at 600 nm wavelength using a spectrophotometer (LKB, Ultrospec II, USA).

In addition, the expression level of G-CSF, together with five purified G-CSF standards of known concentration, SDS-PAGE of the sampled sample, and then stained and detected by a laser scanner, An image analyzer was used to obtain the peak area of the stained portion of the sample by comparing the quantitative curve created using the peak area of the standard material.

The stability of the plasmid was diluted 10 times by successively the sample collected, smeared and cultured in LB plate to form a single colony, and then transferred to an LB plate containing ampicillin at a concentration of 100 µg / ml and cultured. , The percentage of colonies showing double ampicillin resistance.

FIG. 2 is a graph showing cell growth and plasmid stability according to G-CSF protein induction time according to fermentation time. FIG. 3 is a graph showing G-CSF protein expression pattern according to G-CSF protein induction time according to fermentation time. to be. In FIGS. 2 and 3, induction phase 1 represents the case of L-arabinose addition at the logarithmic growth point during DO-stat fed-batch cultivation process, and induction phase 2 represents L-arabinose at the beginning of the incubation. Induction phase 3 represents the case where L-arabinose was added about 4 hours after the start of oil price culture, and induction period 4 indicates L when about 8 hours after the start of oil price culture. The case where arabinose is added is shown.

As shown in FIG. 2 and FIG. 3, when expression is induced at the logarithmic growth point in the initial medium, the expression level of G-CSF remains high as the fermentation progresses, but the stability of the plasmid is sharply lowered later in the fermentation. Could be found; In contrast, G-CSF was not well expressed when protein expression was induced in the latter part of DO-stat feeding (at about 4 and 8 hours after the start of feeding). When protein expression was induced at the beginning of fed-batch culture, not only the expression level of G-CSF was maintained high but also the stability of plasmid was maintained at a high level until the latter part of fermentation.

Therefore, the optimal protein expression induction time for maximizing cell concentration, plasmid stability, and G-CSF expression amount was found to be the point at which the batch culture and the fed-batch culture begin.

Example 3

[Production of G-CSF by DO-stat Fed-Batch Culture Using Glucose as Carbon Source]

Glucose as a carbon source is excellent in terms of growth rate and yield of Escherichia coli, but when present in high concentrations above a certain concentration, it not only strongly inhibits arabinose operon, but also produces a lot of by-product acetic acid, which decreases the yield of protein. have. Based on these reports, in this practice, glycerol was used as the carbon source in the batch fermentation section in which high concentration of glucose was present, and in the DO-stat fed-batch culture, the accumulation of carbon source and the by-products such as acetic acid caused by In the fed-batch fermentation section, glucose was used as a carbon source because it could be prevented. At this time, the glucose concentration in the culture was maintained at 0.5g / L or less. The results of using glucose as a carbon source in the DO-stat fed-batch fermentation section were compared with those of glycerol.

FIG. 4 is a graph showing the cell concentration and plasmid stability of the DO-stat fed-batch culture using glucose or glycerol as a carbon source according to the fermentation time. FIG. 5 is a DO-stat fed-batch culture using glucose or glycerol as a carbon source. It is a graph showing the concentration of glycerol and glucose remaining in the G-CSF and the culture medium at the time of fermentation.

As shown in FIGS. 4 and 5, it was found that the cell concentration, the stability of plasmid and the expression level of G-CSF were not significantly different from those of glycerol as a carbon source. Therefore, it was confirmed that glucose can be used as an excellent carbon source comparable to expensive glycerol in the DO-stat fed-batch culture of the present invention.

As described and demonstrated in detail above, according to the DO-stat fed-batch fermentation method of the present invention, in the batch fermentation section in which carbon sources are present at high concentration, glycerol is used as the carbon source to reduce the inhibitory effect of arabinose operon and accumulation of acetic acid. In the oil price cultivation section using the amount of dissolved oxygen as an indicator, G-CSF expression inhibition was prevented by maintaining the low-cost, high-efficiency glucose as a carbon source below the minimum expression inhibition level. -Arabinose is added to maintain cell growth, plasmid stability, and G-CSF expression level stably at a high level, thereby mass-producing G-CSF at low production cost.

Claims (8)

Recombinant Escherichia coli containing an inducible G-CSF expression vector using L-arabinose operon was batch cultured in an initial culture medium containing a carbon source other than glucose, and then the amount of dissolved oxygen rapidly increased. DO-stat fed-batch culture method for mass production of G-CSF comprising the step of culturing in fed medium, and adding L-arabinose to induce protein expression. The method of claim 1, wherein the recombinant E. coli is E. coli MC1061: pWAGF (KFCC-10961) DO-stat fed-batch culture method. The method according to claim 1, wherein the initial culture medium is a medium consisting of yeast extract 40g / L, glycerol 40g / L, Na ₂ HPO ₄ 10.22g / L, KH ₂ PO ₄ 2.31g / L and ampicillin 0.07g / L DO-stat fed-batch culture method characterized in that. The method of claim 1 wherein the amount of dissolved oxygen is 50

If above, infuse oil badge, 50

DO-stat fed-batch cultivation method characterized in that the stop of the feeding medium fed to fall below. 5. The DO-stat fed-batch culturing method according to claim 1 or 4, wherein the medium fed oil is a medium composed of 292 g / L of yeast extract, 400 g / L of carbon source, and 5.76 g / L of anhydrous magnesium sulfate. 6. The DO-stat fed-batch culture method according to claim 5, wherein the carbon source is glucose. [Claim 7] The DO-stat fed-batch culture method according to claim 1 or 6, wherein when glucose is used as the carbon source of the fed medium, the concentration of glucose is maintained at 0.5 g / L or less during the fed-batch culture. The DO-stat fed-batch culture method according to claim 1, wherein L- aribinose is added at the start of DO-stat fed-batch culture.

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