Method for improving expression quantity of transient transfected recombinant protein of CHO-K1 cell

Abstract

The invention discloses a method for improving the expression quantity of transient transfected recombinant protein of CHO-K1 cells. The method comprises the following steps: S1, recovering the CHO-K1 cells and subculturing the CHO-K1 cells; s2, adding the cells of which the density is adjusted into a recombinant protein DNA-PEI compound to carry out transient transfection; s3, adding an additive into the transfection system, wherein the additive is a mixture of a sodium butyrate solution and an EN3 solution. According to the method, the culture medium, the cell density before transfection and the proportion of recombinant protein DNA to PEI in a transfection system are optimized, and the additive is added after transfection, so that the antibody expression after cell transfection is promoted, and the expression level of the recombinant protein is effectively improved.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a method for improving the expression level of recombinant proteins by transient transfection of CHO-K1 cells. Background Art

[0002] As a core product in the field of biopharmaceuticals, recombinant proteins play an irreplaceable role in disease treatment, vaccine development, diagnostic reagents, and basic scientific research. Mammalian cell expression systems have become the preferred platform for producing complex recombinant proteins due to their ability for post-translational modification of eukaryotic proteins (such as glycosylation, folding, etc.). Among them, Chinese hamster ovary cells (CHO-K1) have become the mainstream host cells for commercial production of recombinant proteins due to their high genetic stability, suspension culture ability, and compliance with industrial scale-up production specifications.

[0003] In the production process of recombinant proteins, transient transfection technology is widely used in small-scale preparation and process optimization in the R & D stage due to its characteristics of not requiring the establishment of stable cell lines and being able to quickly obtain the target product. Transient transfection transiently introduces exogenous plasmid DNA into host cells and uses the intracellular transcription and translation mechanism to transiently express the target protein, usually reaching the expression peak within a few days after transfection. However, there are still significant bottlenecks in the transient transfection efficiency and recombinant protein expression level of CHO-K1 cells. Research shows that the transfection efficiency of traditional transfection methods (such as liposome- or polyethylenimine-mediated transfection) in CHO-K1 cells is usually less than 30%, and the cytotoxicity of high-concentration plasmids or transfection reagents easily leads to cell apoptosis, further reducing protein production. In addition, existing expression vectors are mostly designed for stable transfection, and their promoter activity, secretion signal peptides, and resistance screening markers are not well adapted in the transient expression system, resulting in low transcription efficiency of exogenous genes or limited protein secretion ability.

[0004] In recent years, researchers have tried to improve the transient expression level by optimizing vector elements (such as introducing strong promoters, matrix attachment region MAR elements), developing new transfection reagents (such as cationic polymer nanoparticles), or adjusting culture conditions (such as adding histone deacetylase inhibitors), but these strategies often have defects such as high cost, complex processes, or instability between batches. Therefore, how to improve the efficiency of transient transfection and increase the expression level has become an urgent problem to be solved. Summary of the Invention

[0005] To solve the deficiencies of the prior art, the purpose of the present invention is to provide a method for improving the expression level of recombinant proteins by transient transfection of CHO-K1 cells, which optimizes the culture medium, cell density before transfection, and the ratio of recombinant protein DNA to PEI in the transfection system, and adds additives after transfection to promote antibody expression after transfection of cells, effectively improving the expression level of recombinant proteins.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] A method for improving the expression level of recombinant protein in transient transfection of CHO-K1 cells, comprising the following steps:

[0008] S1. Resuscitate and passage the CHO-K1 cells for culture;

[0009] S2. Add the cells with adjusted density to the recombinant protein DNA-PEI complex for transient transfection;

[0010] S3. Add an additive to the transfection system, and the additive is a mixture of sodium butyrate solution and EN3 solution.

[0011] Preferably, before transient transfection, the cell density is adjusted to 5.5 - 6.5×10 6 cells / mL.

[0012] Preferably, the transient transfection time is 18 - 20 h, and the rotation speed of the shaker is 120 - 200 rpm.

[0013] Preferably, the sodium butyrate solution is a sterile solution with a concentration of 100 mM using PBS buffer as the solvent, and its addition amount is 22 - 26 μL / 1 mL of transfection solution.

[0014] Preferably, the EN3 solution is a sterile mixed solution of glutamine and glucose, and the concentrations of glutamine and glucose in the culture medium are 28 - 30 g / L and 180 - 220 g / L respectively, and its addition amount is 45 - 55 μL / 1 mL of transfection solution.

[0015] Preferably, the recombinant protein DNA-PEI complex comprises recombinant protein DNA and PEI with a mass ratio of 1:4 - 1:6.

[0016] Preferably, before step S1, the CHO-K1 cells are centrifuged and resuspended, with a rotation speed of 700 - 900 rpm and a time of 4 - 6 min.

[0017] Preferably, after the cell density is adjusted, the cell culture conditions are: temperature is 37°C, 5% volume percentage of CO2, and the rotation speed is above 120 rpm.

[0018] Preferably, the TCA cycle enhancer is α-ketoglutaric acid (α-KG), and its concentration in the culture medium is 2 - 4 mM.

[0019] The beneficial effects of the present invention are:

[0020] (1) The present invention optimizes the culture medium, the cell density before transfection, and the ratio of recombinant protein DNA to PEI in the transfection system, and adds additives after transfection to promote the antibody expression after cell transfection, effectively improving the expression level of the recombinant protein.

[0021] (2) The present invention adds α-KG, which can improve the efficient operation of the TCA cycle, further improve the efficient expression of the recombinant protein. α-KG can also promote the conversion of glutamine in the EN3 solution into glutamate through glutaminase and further enter the TCA cycle, forming a "glutamine-α-KG" metabolic axis, significantly enhancing the carbon source utilization rate, thereby improving the efficient operation of the TCA cycle. Specific Embodiments

[0022] The present invention will be specifically introduced below in combination with specific embodiments.

[0023] Example 1. A method for improving the transient transfection recombinant protein expression level of CHO-K1 cells, comprising the following steps:

[0024] S1. Take CHO-K1 cells in the logarithmic growth phase, centrifuge at 800 rpm for 5 minutes, resuspend the cells, and then use the culture medium to passage the cells until the 3rd passage.

[0025] S2. Before transfection, adjust the cell density, and place the cells with adjusted density in a shaker for transfection.

[0026] Prepare a 10 mL recombinant protein DNA-PEI complex transfection system, and the preparation method is as follows:

[0027] (1) Preparation of DNA buffer: Take 100 μg of recombinant protein expression plasmid DNA, add it to 4 mL of fresh culture medium inoculated with passage cells in advance, gently vortex and mix well, and incubate at room temperature for 5 minutes to fully disperse the DNA in the culture medium.

[0028] (2) Preparation of PEI buffer: Weigh 300 μg of polyethyleneimine (PEI) according to the mass ratio of DNA:PEI of 1:3, add it to another 4 mL of fresh culture medium containing passage cells, vortex and oscillate until the reagent is completely dissolved, and incubate at room temperature for 5 minutes to form a homogeneous PEI solution.

[0029] (3) Complex assembly and optimization: Dropwise add the PEI buffer to the DNA buffer, and at the same time supplement the passage culture medium to make the total volume of the system reach 10 mL. After gently pipetting and mixing well, let it stand and react at room temperature for 20 minutes to promote the self-assembly of DNA and PEI through electrostatic interaction to form a stable complex.

[0030] (4) Cell transfection operation

[0031] Take out the CHO-K1 cell suspension with appropriate density from the constant temperature shaker. Fix the bottom of the shaking flask with one hand and slowly rotate it in a fixed direction. Hold the pipette with the other hand and add the DNA-PEI complex drop by drop evenly into the culture system. After the sample addition is completed, immediately return the shaking flask to the constant temperature shaker and maintain the standard culture conditions (37 °C, 5% CO2, 120 rpm) for subsequent protein expression.

[0032] Put the cells into the shaker for culture for 18 - 20 h, add sodium butyrate solution, EN3 solution and TCA cycle enhancer, and continue the culture. After 10 days of culture or when the cell viability is lower than 80%, collect samples, count, and measure the protein concentration after downstream purification.

[0033] Performance detection test

[0034] (1) Evaluate the effects of medium type and cell seeding density on recombinant protein expression in the CHO-K1 cell transient transfection system:

[0035] Three commercial media were selected for the experiment: OPMI OPM-CHO CD08, BBI Rapid TransCHO, and Consen CHO CD04, which were respectively combined with three cell densities of 5.0×10 6 、6.0×10 6 、7.0×10 6 cells / mL for orthogonal optimization. The specific results are shown in Table 1.

[0036] Table 1 Effects of cell density at transfection on recombinant protein expression under different media

[0037]

[0038] As can be seen from Table 1, at the condition of 6.0×10 6 cells / mL, the protein expression levels of all media were significantly higher than those of the other two groups, confirming that cell density is the core parameter affecting transient transfection efficiency. Excessive cell density (7.0×10 6 cells / mL) may lead to nutrient competition or accumulation of metabolic by-products, inhibiting the expression of exogenous genes. When the cell density was 6.0×10 6 cells / mL, the protein yield of BBI Rapid TransCHO medium reached 315 mg / L, and the yields of Consen CHO CD04 and OPMI OPM-CHO CD08 decreased in turn, suggesting that medium components (such as energy substances, growth factors and osmotic pressure regulators) have specific regulatory effects on gene delivery efficiency and cell metabolic status. Based on the above results, this study determined that BBI Rapid TransCHO medium combined with 6.0×10 6The cell density of cells / mL is the optimal condition for the transient transfection process, and this combination will be used as the benchmark parameter for subsequent large-scale production and process verification. This optimization strategy provides a repeatable experimental basis for improving the transient expression efficiency of CHO-K1 cells.

[0039] (2) Effect of the mass ratio of recombinant protein DNA to PEI on the expression level of recombinant protein:

[0040] The experimental procedure was the same as in Example 1. Cells were passaged using BeiGene Rapid TransCHO medium, and the cell density was adjusted to 6.0×10 6 cells / mL. The dosages of polyethyleneimine (PEI) were set at 300 μg, 400 μg, and 500 μg respectively. The specific results are shown in Table 2.

[0041] Table 2 Effect of the mass ratio of recombinant protein DNA to PEI on the expression level of recombinant protein

[0042]

[0043] According to Table 2, when transient transfection was performed using BeiGene Rapid TransCHO medium at a density of 6.0×10 6 cells / mL, when the mass ratio of recombinant protein DNA to polyethyleneimine (PEI) was 1:5, the cell transfection efficiency was significantly improved, and the expression level of recombinant protein reached the peak; when the mass ratios were 1:3 and 1:4, the protein expression levels were similar and no significant difference was shown.

[0044] (3) Effect of adjusting the shaker speed on the expression level of recombinant protein:

[0045] The experimental procedure was the same as in Example 1. Cells were passaged using BeiGene Rapid TransCHO medium, and the cell density was adjusted to 6.0×10 6 cells / mL, and the ratio of recombinant protein DNA:PEI = 1:5. The shaker speeds were set at 90 rpm, 105 rpm, and 120 rpm respectively. The specific results are shown in Table 3.

[0046] Table 3 Effect of cell density on the expression level of recombinant protein during transfection under different media

[0047]

[0048]

[0049] According to Table 3, as the shaker speed increased, the expression level increased, and the expression level was the highest at a speed of 120 rpm.

[0050] (4) Set Comparative Example 1, which is different from Example 1 in that no transient transfection treatment is performed. In Example 1, the cells are passaged using the BBI Life Sciences Rapid TransCHO medium, and the cell density is adjusted to 6.0×10 6 cells / mL, and the protein concentration is measured. The specific results are shown in Table 4.

[0051] Table 4 Expression levels of recombinant proteins in Comparative Example 1 and Example 1

[0052] Comparative Example 1 Example 1 Expression level (mg / L) 356 632

[0053] As can be seen from Table 4, the expression level of the recombinant protein in Example 1 is significantly higher than that in Comparative Example 1.

[0054] (5) Set Comparative Example 2, which is different from Example 1 in that no TCA cycle enhancer is added. In Example 1, the cells are passaged using the BBI Life Sciences Rapid TransCHO medium, and the cell density is adjusted to 6.0×10 6 cells / mL, and the protein concentration is measured. The specific results are shown in Table 5.

[0055] Table 5 Expression levels of recombinant proteins in Comparative Example 2 and Example 1

[0056] Comparative Example 2 Example 1 Expression level (mg / L) 427 632

[0057] As can be seen from Table 4, the expression level of the recombinant protein in Example 1 is significantly higher than that in Comparative Example 2. This is because α-ketoglutaric acid (α-KG) is the product of the reaction catalyzed by isocitrate dehydrogenase in the TCA cycle and directly participates in the subsequent step of conversion to succinyl coenzyme A. Adding α-KG can rapidly increase its intracellular concentration, accelerating the overall operation rate of the cycle. Moreover, α-KG can also promote the conversion of glutamine in the EN3 solution to glutamate through glutaminase and further enter the TCA cycle, forming a "glutamine-α-KG" metabolic axis, significantly improving the carbon source utilization rate, thereby enhancing the efficient operation of the TCA cycle and further increasing the efficient expression of the recombinant protein.

[0058] The above shows and describes the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any form. Any technical solutions obtained by means of equivalent replacement or equivalent transformation fall within the protection scope of the present invention.

Claims

1. A method for improving the expression level of recombinant proteins by transient transfection of CHO-K1 cells, characterized in that, It includes the following steps: S1. Resuscitate and subculture CHO-K1 cells; S2. Add cells with adjusted density to the recombinant protein DNA-PEI complex for transient transfection; S3. Add an additive to the transfection system, and the additive is a mixture of sodium butyrate solution, EN3 solution and TCA cycle synergist.

2. The method for increasing the expression level of recombinant protein in transient transfection of CHO-K1 cells according to claim 1, wherein Before step S1, the CHO-K1 cells are centrifuged and resuspended, with the rotation speed of 700-900 rpm and the time of 4-6 min.

3. The method for increasing the expression level of recombinant protein by transient transfection of CHO-K1 cells according to claim 1, characterized in that, The recombinant protein DNA-PEI complex includes recombinant protein DNA and PEI with a mass ratio of 1:4-1:

6.

4. The method for increasing the expression level of recombinant protein by transient transfection of CHO-K1 cells according to claim 1, characterized in that Adjust the cell density to 5.5~6.5×10 6 cells / mL before transient transfection.

5. The method for increasing the expression level of recombinant protein in transient transfection of CHO-K1 cells according to claim 1, characterized in that, The transient transfection time is 18-20 h, and the rotation speed of the shaker is 120-200 rpm.

6. The method for increasing the expression level of recombinant protein by transient transfection of CHO-K1 cells according to claim 1, characterized in that, The sodium butyrate solution is a sterile solution with a concentration of 100 mM using PBS buffer as the solvent, and its addition amount is 22-26 μL / 1 mL transfection solution.

7. The method for increasing the expression level of transiently transfected recombinant protein in CHO-K1 cells according to claim 1, wherein The EN3 solution is a sterile mixed solution of glutamine and glucose, and the concentrations of glutamine and glucose in the culture medium are 28-30 g / L and 180-220 g / L respectively, and its addition amount is 45-55 μL / 1 mL transfection solution.

8. The method for increasing the expression level of recombinant protein by transient transfection of CHO-K1 cells according to claim 1, wherein The TCA cycle synergist is α-ketoglutaric acid, and its concentration in the culture medium is 2-4 mM.

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