TW201900879A - Cell culture method at low temperature applicable to mass production of protein - Google Patents

Abstract

The present disclosure relates to a method useful in mass production of a protein using microorganisms, by increasing the concentration of a nitrogen source at a low temperature of higher than or equal to 20 DEG C but lower than 30 DEG C. The method according to the present disclosure is useful for the expression of an active protein in high yield, which is suitably applicable to mass production while suppressing the production of unnecessary insoluble proteins from microorganisms.

Description

 Cell culture method capable of mass-producing protein at low temperature  

The present disclosure relates to a method for producing a protein by increasing the concentration of a nitrogen source at a low temperature of 20 ° C or higher but lower than 30 ° C using a microorganism.

The current method of producing recombinant proteins by microorganisms causes the formation or agglomeration of major improper folding of the protein, especially when the recombinant protein is a protein derived from an animal cell, and as a result, inactive inclusion bodies are often produced. In addition, one of the problems with this method is the follow-up work, such as the need to refold in order to use the protein, etc., which is very inefficient.

In order to overcome this problem and increase the production of appropriately folded active proteins, many research experiments have been conducted. Basically, in order to produce an active protein from a microorganism, it is necessary to insert a signal gene to induce movement to a foldable position. Traditionally, attempts have been made to target proteins with an accompanying protein that prevents the formation of inclusion bodies and assists in folding (Korean Patent Case No. 10-2009-0114422); the promoter is changed by changing the inducing conditions (Champion et al. 2001) Proteomics , 1:1133-1148); or changing the fermentation conditions (Schein (1989) Bio/Technology , 7: 1141-1149).

In addition, it has also been attempted to reduce the rate of protein production by using performance conditions lower than or equal to 30 ° C to suppress the production of insoluble proteins. However, since the optimum growth temperature of a general microorganism is from 33 ° C to 37 ° C, a low temperature condition of less than or equal to 30 ° C inevitably has a problem of a decrease in the growth potential of the microorganism, and as a result, the amount of the target protein in the microorganism is greatly reduced. Thus, the low yield of such products is extremely lethal to the use of microorganisms to produce physical properties in quantities.

In order to solve this problem, studies such as changing the promoter into a low temperature resistant gene (Vasina and Baneyx (1996) Appl. Environ. Microbiol, 1444-1447) and the like are being carried out. However, this method has a serious disadvantage in that it always requires the use of a low temperature resistant promoter, and the amount of target protein varies depending on the combination of the target protein and the promoter, and thus the method cannot be applied to all protein types that are industrially or widely used.

On the other hand, attempts have been made to increase the yield of a target protein derived from a microorganism by adjusting a component in the medium. However, it has been reported that when excessive nitrogen sources are introduced, there is a risk of reducing growth potential and protein performance (Popplewell et al. (2005) Methods in Molecular Biology, 308: 17-30).

Under such circumstances, the present inventors have made efforts to increase protein production, and have completed the present disclosure by finding a method for preparing active protein in high yield, which is suitable for application in mass production, while suppressing the generation of unnecessary microorganisms. Insoluble protein.

One of the objects of the present disclosure is to provide a method for preparing a protein by microbial fermentation, wherein the method comprises: (a) fermenting a microorganism containing the recombinant vector at a temperature higher than or equal to 20 ° C but lower than or equal to 30 ° C. 48 hours or longer; and (b) adding an N-containing medium before or during the start of step (a) microbial fermentation.

The disclosure will be described in more detail below.

On the other hand, the description and embodiments of the disclosure are applicable to other descriptions and embodiments, respectively. That is, all combinations of the various elements disclosed in the disclosure are within the scope of the disclosure. In addition, the following explicit description should not be construed as limiting the scope of the disclosure.

In addition, many of the equivalents of the disclosed embodiments of the present disclosure can be understood or appreciated by those skilled in the art. Moreover, such equivalents are intended to be included in this disclosure.

In order to achieve the above object, a method of the present invention is a method for preparing a protein by microbial fermentation, wherein the method comprises: (a) the microorganism comprising the recombinant vector is higher than or equal to 20 ° C but lower than or equal to 30 ° C. The temperature is fermented for 48 hours or longer; and (b) the medium containing the N-source is added before or during the start of the step (a) microbial fermentation.

Typically, high temperature conditions or temperature shifting methods are attempted to accelerate growth while reducing impurities in the production of proteins, and low temperature conditions are generally not attempted because such conditions are too harsh for growth. However, the importance of the present disclosure is that the inventors have first discovered that by adding an excessive amount of nitrogen source under low temperature conditions, protein yield can be increased not only at the laboratory scale but also under mass production.

Each step of the method of preparing a protein by microbial fermentation will be described in detail. The first step (a) is to ferment the microorganism containing the recombinant vector for 48 hours or longer at a temperature higher than or equal to 20 ° C but lower than or equal to 30 ° C.

In the present disclosure, the microorganism for fermentation is not limited as long as it includes a recombinant vector which can express the target protein. In addition, one of ordinary skill in the art can select and apply microorganisms containing appropriate recombinant vectors depending on the purpose.

The term "microorganism comprising a recombinant vector" as used herein refers to a microorganism transformed by a vector having a gene encoding one or more proteins of interest.

Furthermore, the term "vector" as used herein, refers to a DNA product comprising a nucleotide sequence encoding a polynucleotide of a protein of interest operably linked to a suitable regulatory sequence such that the protein of interest is expressed in a suitable host. Regulatory sequences can include promoters that initiate transcription, any sequence of sequences that regulate such transcription, sequences that encode suitable mRNA ribosome-binding sites, and sequences that regulate termination of transcription and translation. Upon transformation into a suitable host, the vector may replicate or perform its function, unaffected by the host genome, or may integrate into the genome itself.

In the present disclosure, any vector which can be replicated in a host cell can be used without any particular limitation as long as it is known in the art. Examples of commonly used vectors are plastids, vesicles, viruses, and phage in a natural or recombinant state. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used as phage vectors or viscous carriers, and pBR systems, pUC systems, pBluescript II systems, pGEM systems, pTZ systems. The pCL system, and the pET system can be used as a plastid carrier. Specifically, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, and pCC1BAC vectors can be used, but are not limited thereto.

The carrier which can be used in the present disclosure is not particularly limited, and a known expression carrier can also be used. Further, a polynucleotide encoding a target protein can be inserted into a chromosome using a vector for insertion of a chromosome in a cell. The polynucleotide to chromosome insertion method can be carried out by any method known in the art, for example, homologous recombination, but is not limited thereto. The vector of the present disclosure may further comprise a selection marker for insertion of the indicator vector into the chromosome. The selection marker is used to select for cells transformed by the vector; that is, the marker is used to confirm whether the target nucleic acid molecule has been inserted. Thus, markers that confer a selectable phenotype (eg, drug resistance, nutritional requirements, resistance to cytotoxic agents, or surface protein performance) can be used. Only cells exhibiting a selectable marker can survive or display different phenotypes in an environment treated with a selective agent, thus allowing for the selection of transformed cells.

The term "selectable marker" as used herein refers to a gene having antibiotic resistance. In addition, cells having these genes can survive even in an antibiotic-treated environment, and thus can be effectively used as a selection marker in the process of obtaining a large number of plastids in E. coli . Since the antibiotic resistance gene is not an element which greatly affects the performance efficiency in the optimized recombination method which is the core technology of the present disclosure, the antibiotic resistance gene which is commonly used as a selection marker can be used without limitation. For example, it is clear that it can be resistant to ampicillin, tetracycline, kanamycin, chloramphenicol, streptomycin, or neomycin. Genes, more specifically, genes that are resistant to tetracycline can be used, but are not limited by this.

The term "transformation" as used herein refers to the introduction of a vector comprising a polynucleotide encoding a protein of interest into a host cell to express the protein encoded by the polynucleotide in the host cell. As long as the transformed polynucleotide can be expressed in a host cell, it can be inserted into and placed in the chromosome of the host cell or present extrachromosomally. Further, the polynucleotide includes DNA and RNA encoding the protein of interest. The polynucleotide may be introduced in any form as long as it can be introduced into and expressed in a host cell. For example, a polynucleotide can be introduced into a host cell in a sputum-type format, which is a genetic construct including all of the elements required for its autonomous expression. Typically, expression 匣 can include a promoter operably linked to a polynucleotide, a transcription termination signal, a ribosome binding site, or a translation termination signal. Performance 匣 can be self-replicating performance carrier type. In addition, the polynucleotide may be introduced into the host cell in its own form, operably linked to the sequence required for expression in the host cell, but is not limited thereto. Transformation methods can include any method of introducing a nucleic acid into a cell, but can be carried out according to the host cell, using suitable standard techniques known in the art. For example, suitable standard techniques may be selected from electroporation, calcium phosphate (Ca(H ₂ PO ₄ ) ₂ , CaHPO ₄ , or Ca ₃ (PO ₄ ) ₂ ) precipitation, calcium chloride (CaCl ₂ ) precipitation, Microinjection, polyethylene glycol (PEG) technology, DEAE-dextran technology, cationic liposome technology, and lithium acetate-DMSO technology, but are not limited by this.

Furthermore, the term "operably linked" as used herein, refers to a functional linkage between a polynucleotide sequence encoding a protein of interest and a promoter sequence that initiates and mediates a polynucleotide encoding a target protein of interest. Operable ligation can be made using genetic recombination techniques known in the art. In addition, site-specific DNA cleavage and ligation can be made using dissociating enzymes and ligases known in the art, without being limited by this.

In addition, the term "production" as used herein means that the amount of protein produced is industrially usable. For example, mass production means that the protein produced can be used on a 5L scale or higher, a 30L scale or higher, and a 50L scale or higher.

Specifically, the microorganism containing the recombinant vector can be fermented for 48 hours or longer at a temperature higher than or equal to 20 ° C but lower than or equal to 30 ° C, but is not limited thereto. A temperature higher than or equal to 20 ° C but lower than or equal to 30 ° C belongs to the low temperature in the general fermentation step, and the low temperature fermentation is the newly determined fermentation temperature of the inventors.

The temperature of such low temperature fermentations includes temperatures above about 20 ° C but below or equal to 30 ° C. The term "about" as used herein means all ranges including ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and such ranges include all equal or similar values that appear after the term "about". However, the term is not particularly limited.

Low temperature may mean higher than or equal to 20 ° C but lower than or equal to 30 ° C, or higher than 20 ° C but lower than 30 ° C. For example, the low temperature may be higher than or equal to 21 ° C but lower than or equal to 29 ° C, or higher than 21 ° C but lower than 29 ° C; higher than or equal to 22 ° C but lower than or equal to 28 ° C, or higher than 22 ° C but Below 28 ° C; higher than or equal to 23 ° C but lower than or equal to 27 ° C, or higher than 23 ° C but lower than 27 ° C; higher than or equal to 24 ° C but lower than or equal to 26 ° C, or higher than 24 ° C but Below 26 ° C; or about 25 ° C, but not limited by this.

In addition, the fermentation time can be 48 hours or longer but 72 hours or less.

In the method of preparing a protein by microbial fermentation, the step (b) is a step of adding a medium containing a nitrogen source before or during the step of (a) microbial fermentation.

Specifically, the addition of the medium containing the nitrogen source in the step (b) can be varied depending on the culture method in the art. Specifically, the nitrogen-containing source medium can be added before or during the start of microbial fermentation.

Further, in addition to batch culture, the culture may also be fed-batch culture, but is not limited thereto.

Specifically, the medium containing the nitrogen source before the start of microbial fermentation is inoculated in a medium containing all the nutrients required for the growth or substance production of the bacteria in the batch culture to culture the microorganisms, so it is difficult to add during the cultivation. New medium. In the present disclosure, it may include using a batch culture to inoculate a microbial fermentation containing a recombinant vector in a nitrogen-containing source medium.

In addition, the nitrogen-containing source medium is added during the microbial fermentation to feed-batch culture, which will be contained immediately after the start of fermentation or after the cells have grown to a predetermined stage, or when the nutrient has been consumed in the medium. A medium of one or more nutrients is continuously fed into the culture. In feed-batch culture, a single nutrient or carbon source is usually used as a growth limiting factor. Other nutrients can also be used as a limiting factor. For example, a nitrogen source, oxygen, sulfur, phosphorus, or the like can be used as a limiting factor. The present disclosure includes a medium for continuously supplying a nitrogen-containing source when fermented by a microorganism containing a recombinant vector.

In the present disclosure, the medium includes a medium for culturing cells, a medium for maximizing production of a target protein, and the like.

In particular, the term "cell culture medium" or "medium" refers to a nutrient solution used to maintain, grow, proliferate, or amplify cells in an artificially in vitro environment, which is external to a multicellular organism or tissue. The cell culture medium can be optimized for culturing specific cells, for example, a basal medium prepared for supporting cell growth, a production medium prepared for promoting production of a target protein, and a concentrated medium prepared by concentrating nutrients at a high concentration. . The term nutrient and medium component means a component constituting a medium, which may be used interchangeably in the present disclosure.

Specifically, the term "basal culture medium or basal medium" refers to a medium that can support the basic growth of cells. The basal medium refers to a medium that provides vitamins and minerals in addition to carbon sources and nitrogen sources.

Further, specifically, the term "cell culture production medium" or "production medium" refers to a medium for optimizing the performance of a target protein in a bioreactor. The composition of the production medium may be the same as or different from the basic medium, and when not different, the medium may be produced by concentrating the basal medium or adding a specific component to the basal medium.

Further, the "feeding medium" and the "addition medium" may be a medium composed of a specific nutrient or a plurality of nutrients, both of which are concentrated components of the basal medium, but are not limited thereto. In addition, the components and concentrations of the feed medium may vary depending on the cells cultured by those of ordinary skill in the art.

Further, in the present disclosure, when all the sugar in the medium is used as the cells grow during the fermentation, the fermentation time can be increased by additionally adding sugar. At this time, the nitrogen source is added in the range of 10 g/L to 80 g/L, which is clearly in the range of 15 g/L to 70 g/L, more specifically in the range of 20 g/L to 64.5 g/L. Within, but not subject to this limitation.

Further, the medium containing the nitrogen source may be a medium containing no substance derived from the animal, but is not limited thereto. More specifically, the medium containing the nitrogen source may include one or more components selected from the group consisting of ammonium sulfate, yeast extract, or a combination thereof, without being limited thereto.

Typical concentrations of nitrogen source in the medium range from 2 g/L to 14 g/L, but excess nitrogen source concentration can be introduced into the medium. Specifically, the nitrogen source concentration can range from 30 g/L to 150 g/L, more specifically from 40 g/L to 120 g/L, more specifically from 49.6 g/L to 106.7 g/L. Within the scope, but not limited to this.

Further, the mass ratio of the nitrogen source to the carbon source is 1:0.15 to 1.24, but is not limited thereto.

In the present disclosure, a larger range of nitrogen sources can be introduced than the general method, and insoluble proteins and inclusion bodies produced by microorganisms at low temperature fermentation can be suppressed to restore the growth potential of microorganisms at low temperatures and generate a large amount of protein for protein. Used on an industrial scale.

The microorganism which can improve the growth or proliferation of microorganisms in the medium containing the nitrogen source can be Escherichia coli , but is not limited thereto.

The term "protein" as used herein refers to a protein expressed in a microorganism that is genetically engineered to express a protein. The protein may be identical or similar to a protein normally expressed in a microorganism, and includes a recombinant protein. In addition, proteins may be heterologous to proteins that are normally expressed in microorganisms. Alternatively, the protein may be a chimeric, a portion of which contains the same or similar amino acid sequence as the protein normally expressed in the microorganism, and the other portion is the exogenous substance of the microorganism. In the present disclosure, the protein may be an antibody or a fragment thereof, but is not particularly limited. The disclosure includes all proteins made using the microorganisms comprising the recombinant vector using the methods of the present disclosure.

In general, in the production of a protein containing a recombinant vector, it is possible to mix various proteins such as an active protein, a truncated protein, an inactive protein, a protein agglutination or the like. However, in the present disclosure, a protein may refer to an active protein, and the active protein may refer to a protein having an active form, and may specifically refer to an L-H protein.

Specifically, the protein disclosed herein may be an antibody, more specifically, a monoclonal antibody.

The term "monoclonal antibody" as used herein refers to an antibody which can be formed by a cell which forms a single antibody and which recognizes an antigenic determinant. The antibodies of the present disclosure may include, but are not limited to, all of the medical antibodies commonly used in the art.

Furthermore, antibodies encompass conceptually full length antibodies and antibody fragments, and examples of antibody fragments include all Fv, Fab, Fab', F(ab') ₂ , Fd, and the like. Fv includes two types of disulfide stabilized Fv (dsFv) and single chain Fv (scFv). Fd refers to the heavy chain component included in the Fab.

The protein prepared by the method of the present disclosure can be used as a medical protein. We use "medical proteins", which are commonly used in biomedical sciences and have various physiological activities. Physiological activities include those whose function is to modulate genetic and physiological functions and to correct abnormal conditions caused by lack or excessive secretion of substances involved in in vivo regulatory functions. Physiological activities can include general protein medical agents.

In the present disclosure, a medical protein may be included without limitation as long as it has physiological activity in vivo. For example, medical proteins can be insulin, insulinotropic peptide, growth hormone (GH), prolactin (LTH), follicle stimulating hormone ( FSH ), factor VIII (factor VIII), factor VII (factor VII), Erythropoietin, adiponectin, antibody, antibody fragment scFv, Fab, Fab', or F(ab') ₂ .

The method for producing a protein according to the present disclosure can be effectively used to express an active protein in a high yield, which is suitable for use in mass production while suppressing the production of unnecessary insoluble proteins from microorganisms.

Figure 1 is a graph showing the results of measuring the growth rate of bacterial cells at each temperature.

Figure 2 shows the SDS-PAGE and Western blot results at each temperature.

Figure 3 is a graph showing the ratio of active protein expression per cell at each temperature.

Figure 4 is a graph showing the growth rate of bacterial cells with nitrogen source concentration at low temperature (25 ° C).

Figure 5 shows the results of low temperature, SDS-PAGE and Western blot changes with nitrogen source concentration.

Figure 6 is a graph showing the results of the increase in the amount of active protein and the percentage (%) of active protein relative to all proteins at low temperatures as a function of nitrogen source concentration.

Figure 7 is a graph showing the results of measuring the growth rate of bacterial cells.

Hereinafter, the disclosure will be described in detail in conjunction with the embodiments. However, the embodiments disclosed herein are for illustrative purposes only and should not be construed as limiting the scope of the disclosure.

Since the various materials required to prepare the disclosed media are available in a variety of different suppliers, materials from suppliers can be used and purchased as needed. Reagent suppliers are companies such as Merck, Sigma-Aldrich, Wako, BD, Dae-Jung, etc., but suppliers are not subject to these restrictions. Further, the ratio of the composition may be appropriately modified in accordance with the embodiment of the present disclosure.

Experimental Example 1: Producing a flask-scale protein

Experiments were carried out according to the following procedures to confirm the protein expression pattern according to temperature at the flask scale. The control of the control group was carried out at 37 ° C, which is known to be suitable for the temperature at which microorganisms are grown. In addition, each experimental group was tested at 18 ° C, 25 ° C, and 33 ° C, and other conditions were the same as in Table 2 below. The fermentation was carried out according to a batch culture process, and the absorbance at 562 nm was measured to confirm the growth potential. When 70% absorbance relative to the maximum absorbance was reached, the fermentation broth was taken and each protein amount was determined by ELISA.

As a result, it was confirmed that in the batch culture process at 18 ° C and 25 ° C culture temperature, the microorganisms maintained extremely low growth even after 24 hours from the start of the culture (Fig. 1). Based on the above results, when the culture temperature is 25 ° C or lower, the growth rate has been found to be remarkably lowered, and the growth rate varies depending on the temperature.

Further, from the results of Figs. 2 and 3, it was confirmed that the ratio of the insoluble protein measured at a temperature of 20 ° C or higher but lower than 30 ° C was drastically decreased, and the active protein was increased.

Experimental Example 2: Protein production in a fermentation tank (5L)

The experiment was carried out on a 5 L scale according to the following procedures to confirm the protein expression pattern depending on the nitrogen source amount in the low temperature culture. Specifically, at a nitrogen source introduction amount of 49.6 g/L to 106.7 g/L, the protein expression is confirmed after the temperature is set to be higher than or equal to 20 ° C but lower than 30 ° C for 40 hours or more after fermentation. degree. The fermentation method was carried out in a feed-batch culture, and the absorbance at 562 nm was measured to confirm the growth potential. When 70% absorbance relative to the maximum absorbance was reached, the fermentation broth was taken and each protein amount was determined by ELISA.

As a result, it was confirmed that the growth rate of the low temperature increased as the concentration of the nitrogen source increased, so the time to reach the maximum cell number was shortened, and the maximum number of cells was also increased (Fig. 4). In addition, it has also been confirmed that the growth potential of low temperature reduction can be overcome by adding an excessive nitrogen source (Fig. 6).

These results suggest that when traditional microorganisms are used to produce enzymes or medical proteins, such as low temperature fermentation and fermentation conditions with excessive nitrogen sources, the low rate of production can be overcome.

Experimental Example 3: Protein production in the fermentation tank (50L)

The nitrogen source was fixed at 74.8 g/L and fermented in a fed-batch culture. In addition, the absorbance at 562 nm was measured to confirm the growth potential. When 70% absorbance relative to the maximum absorbance was reached, the fermentation broth was taken and each protein amount was determined by ELISA.

Since the growth potential of the two batches is very similar, the reproducibility of the fermentation method was confirmed (Fig. 7). Further, it was confirmed by ELISA measurement that the target protein (200 mg/L per batch) was obtained (Table 4). These results confirm that the process can be industrially applicable, that is, patents can be obtained.

It will be apparent to those skilled in the art that the present disclosure may be practiced in other specific forms without departing from the spirit and scope of the invention. Therefore, the embodiments disclosed herein are for illustrative purposes only and are not intended to limit the scope of the disclosure. Rather, the invention is not limited to the embodiments of the present invention, and is intended to cover various alternatives, modifications, equivalents, and other embodiments, which may be included in the nature and scope of the disclosure as defined by the appended claims. .

Claims (8)

 A method for preparing a protein by microbial fermentation, wherein the method comprises: (a) fermenting a microorganism comprising the recombinant vector at a temperature higher than or equal to 20 ° C but lower than or equal to 30 ° C for 48 hours or longer; and (b) The medium containing the N-source is added before or during the start of the step (a) microbial fermentation.    The method of claim 1, wherein the N-containing medium comprises one or more components selected from the group consisting of ammonium sulfate, yeast extract, or a combination thereof.    The method of claim 1, wherein the N-containing medium does not include a substance derived from an animal.    The method of claim 1, wherein the N-source concentration is from 49.6 g/L to 106.7 g/L.    The method of claim 1, wherein the mass ratio of the N-source to the carbon source is 1:0.15 to 1.24.   The method of claim 1, wherein the microorganism is Escherichia coli .  The method of claim 1, wherein the protein is an antibody or a fragment thereof.    The method of claim 1, wherein the protein is a medical protein.