Method for improving expression quantity of HEK 293 cell transient transfection recombinant protein

Abstract

The invention belongs to the technical field of biology, and relates to a method for improving the expression quantity of HEK 293 cell transient transfection recombinant protein, which comprises the following steps: resuscitating and subculturing HEK 293 cells, adding the cells with adjusted density into a recombinant protein DNA-PEI compound to carry out first transient transfection, and carrying out second transient transfection to obtain the HEK 293 cell transient transfection recombinant protein. The HEK 293 cells are subjected to secondary transient transfection after the primary transient transfection, an additive is added into a transfection system after the secondary transient transfection, and the additive comprises ammonium ferric citrate and tryptone. According to the method, the expression level of the target protein is remarkably improved.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a method for increasing the expression amount of a recombinant protein in transient transfection of HEK 293 cells. Background Art

[0002] Recombinant proteins are artificially produced by combining genetic materials from different sources. Recombination refers to the recombining or connecting genetic materials to create new combinations that do not occur naturally. Recombinant proteins used clinically include recombinant hormones, interferons, interleukins, growth factors, tumor necrosis factors, coagulation factors, etc., which are used to treat major diseases such as diabetes, dwarfism, and myocardial infarction. Recombinant proteins are an important aspect of modern biotechnology and are widely used in the development of therapeutic drugs, the production of enzymes for industrial processes, and genetically modified organisms for agricultural purposes.

[0003] Transfection is the process by which eukaryotic cells acquire new phenotypes by actively or passively introducing exogenous DNA fragments. Conventional transfection techniques can be divided into two categories, one is transient transfection and the other is stable transfection. In the former, exogenous DNA / RNA is not integrated into the host chromosome, so multiple copies can exist in a host cell, resulting in high levels of expression, but usually only lasts for a few days; the latter is also called permanent transfection, in which exogenous DNA can be integrated into the host chromosome or exist as an episome, but it usually takes several months to complete, which consumes a lot of time and cost.

[0004] HEK293 cells, also known as human embryonic kidney cells, have human post-translational modification capabilities and have the advantages of rapid division and expression in suspended growth in serum-free medium. HEK293 cells can be used to express recombinant proteins, including antibodies, cytokines, etc. by transfecting exogenous genes. They are widely used in biological experiments on target protein expression, making HEK293 cells an important tool in protein expression systems. However, the current expression method of recombinant protein by transient transfection of HEK 293 cells has problems such as low transfection efficiency and low plasmid integration rate, resulting in low recombinant protein expression levels. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a method for increasing the expression amount of recombinant protein in transiently transfected HEK 293 cells. The method of the present invention can significantly increase the expression level of the target protein.

[0006] The technical solution provided by the present invention is as follows:

[0007] A method for improving the expression amount of a recombinant protein in transient transfection of HEK 293 cells, the method comprising the following steps: after HEK 293 cells are revived and subcultured, cells with adjusted density are added to a recombinant protein DNA-PEI complex for a first transient transfection, after the first transient transfection, the HEK 293 cells are subjected to a second transient transfection, and after the second transient transfection, additives are added to the transfection system, wherein the additives include ammonium ferric citrate and trypsin.

[0008] Furthermore, the cell density was adjusted to 2.5-3.5×10 6 cells / mL.

[0009] Furthermore, HEK 293 cells were subjected to a second transient transfection 24 h to 48 h after the first transient transfection.

[0010] Furthermore, the additives were added within 24 hours after the second transient transfection.

[0011] Furthermore, after adding the additives, the final concentration of ammonium ferric citrate in the culture medium is 3-8 μmol / L, and the final concentration of trypsin in the culture medium is 150-250 g / L.

[0012] Furthermore, the recombinant protein DNA-PEI complex includes recombinant protein DNA and PEI, and the mass ratio of the recombinant protein DNA to PEI is 1:4-6.

[0013] Furthermore, the rotation speed of the shaking table during the first transient transfection and the second transient transfection was above 120 rpm.

[0014] Furthermore, the method for adjusting the cell density before the first transient transfection includes: taking HEK 293 cells in the logarithmic growth phase for subculture, and after the third generation, adjusting the cell density for the first time according to 0.7~0.8×10 6 Cells / mL were inoculated and cultured for 20-28 h. The cell density was adjusted for the second time to 2.5-3.5×10 6 cells / mL.

[0015] Furthermore, before subculturing HEK 293 cells, the cells were resuspended by centrifugation at 700-900 rpm for 4-6 min.

[0016] Furthermore, after the first cell density adjustment, the cell culture conditions were: temperature of 37° C., 5% volume percent CO 2 , and a rotation speed of more than 120 rpm.

[0017] Beneficial Effects

[0018] In view of the problem of low expression amount in transient transfection in the prior art, 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, increases the secondary transient transfection, adjusts the shaking speed, and adds ammonium ferric citrate with a final concentration of 5 μmol / L and trypsin with 200 g / L 24 hours after transfection, thereby increasing the expression level of the recombinant protein by nearly 1 times. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a comparison chart before and after transfection scheme optimization. DETAILED DESCRIPTION

[0020] The present invention is described in detail below through specific embodiments.

[0021] Example 1

[0022] In this example, HEK 293 cells in the logarithmic growth phase were centrifuged at 800 rpm for 5 min to resuspend the cells. The cells were then passaged using OPM-293 CD05 medium (Cat. No.: 81075-001), Transpro CD01 medium (MS002-1L-02), and Wayne 293 transfection medium (A21501). After the third passage, that is, 24 h before the first transient transfection of cells, the first cell density adjustment was performed, at 0.5 × 10 6 cells / mL、0.75×10 6 cells / mL, 1.0×10 6 Cells / mL were inoculated into 30 mL of cells in a 125 mL shake flask.

[0023] The inoculated 125 mL shake flask was placed in an incubator at 37°C, 5% CO2, and 90 rpm for 24 h. Samples were taken and counted. The cell viability was observed to be above 95%. The second cell density adjustment was performed. The cell density was adjusted to 2.0×10 using OPM-293 CD05 Medium, Transpro CD01 Medium, and Wayne 293 Transfection Medium, respectively. 6 cells / mL、3.0×10 6 cells / mL、4.0×10 6 cells / mL, and the cells with adjusted density were placed in a shaker for transfection.

[0024] Prepare 30mL of recombinant protein DNA-PEI complex transfection system as follows:

[0025] Add 30 μg of recombinant protein DNA to 900 μL of the above culture medium for the corresponding subculture to obtain DNA buffer, mix thoroughly and let stand for 5 min;

[0026] Add 90 μg PEI (polyethyleneimine) (so that the mass ratio of DNA to PEI is 1:3) to 900 μL of culture medium for the corresponding subculture to obtain PEI buffer, mix thoroughly and let stand for 5 min;

[0027] The PEI buffer was added to the DNA buffer, and then added to the above culture medium of the corresponding subculture inoculation, the volume was fixed to 30 mL, and allowed to stand for 10 minutes to react to obtain the recombinant protein DNA-PEI complex.

[0028] Take the cells with adjusted density out of the shaker, shake the flask gently in the same direction by hand, slowly add the recombinant protein DNA-PEI complex, and continue culturing in the shaker.

[0029] After 6 days of culture or when the cell viability is less than 50%, samples are collected, counted, and the protein concentration is measured after downstream purification.

[0030] Table 1

[0031]

[0032] HEK293 cells were transiently transfected using three culture media (Kangsheng, Opim&Dongning) and three cell expression densities (2.0×10 6 , 3.0×10 6 4.0×10 6 cells / mL) were used for comparison. The three culture media were transfected at a density of 3.0×10 6 cells / mL, the expression level was the highest. Among them, Kangsheng Wayne293 transfection medium was at 3.0×10 6 cells / mL, the highest expression level was 315 mg / L; OPM-293 CD05 medium was second, and the lowest expression level was in Transpro CD01 medium, with a transfection density of 3.0×10 6 cells / mL, the cell density and transfection expression level were higher than 4.0×10 6 cells / mL, it can be seen that cell density is very important for the expression of recombinant proteins, and cell density must be strictly controlled during cell transfection. The optimal screening results of this example will serve as the basis for subsequent experiments, that is, using Kangsheng Wayne293 transfection medium at a density of 3.0×10 6 The first transient transfection was performed at 144 cells / mL.

[0033] Example 2

[0034] Effect of the mass ratio of recombinant protein DNA to PEI on the expression of recombinant protein

[0035] HEK 293 cells in the logarithmic growth phase were centrifuged at 800 rpm for 5 min to resuspend the cells. Then, the cells were passaged using Kangsheng Wayne 293 transfection medium. After the third passage, that is, 24 h before the first transient transfection of the cells, the first cell density adjustment was performed at 0.75 × 10 6 Cells / mL were inoculated into 30 mL of cells in a 125 mL shake flask.

[0036] The inoculated 125 mL shake flask was placed in an incubator at 37°C, 5% CO2, and 90 rpm for 24 h. Samples were taken and counted. The cell status and viability were observed to be above 95%. The second cell density adjustment was performed and the cell density was adjusted to 3.0×10 using Kangsheng Wayne293 transfection medium. 6 cells / mL, and put the cells with adjusted density into a shaker for the first transient transfection.

[0037] Prepare 30mL of recombinant protein DNA-PEI complex transfection system as follows:

[0038] Add 30 μg of recombinant protein DNA into 900 μL Kangsheng Wayne293 transfection medium to obtain DNA buffer, mix thoroughly and let stand for 5 minutes;

[0039] The dosage of PEI was 90 μg, 120 μg, and 150 μg, respectively, which were added to 900 μL of Kangsheng Wayne293 transfection medium to obtain PEI buffer, mixed thoroughly, and allowed to stand for 5 min;

[0040] Add PEI buffer to DNA buffer, then add Kangsheng Wayne293 transfection medium, make the volume to 30mL, let it stand for 10 minutes to react and obtain the recombinant protein DNA-PEI complex.

[0041] Take the cells with adjusted density out of the shaker, shake the flask gently in the same direction by hand, slowly add the recombinant protein DNA-PEI complex, and continue culturing in the shaker.

[0042] After 6 days of culture, or when the cell viability is less than 50%, samples are collected, counted, and the protein concentration is measured after downstream purification.

[0043] Table 2

[0044]

[0045] According to Table 2, the transfection medium of Kangsheng Wayne293 was used at a density of 3.0×10 6 When the mass ratio of recombinant protein DNA to PEI was 1:5, the transfection efficiency was the best and the expression level of recombinant protein was the highest. When the mass ratio of recombinant protein DNA to PEI was 1:3 and 1:4, the expression level had little difference.

[0046] Example 3

[0047] Perform a second transient transfection on the transfected cells to adjust the shaker speed and see the effect on the expression of the recombinant protein

[0048] HEK 293 cells in the logarithmic growth phase were centrifuged at 800 rpm for 5 min to resuspend the cells. Then, the cells were passaged using Kangsheng Wayne 293 transfection medium. After the third passage, that is, 24 h before the first transient transfection of the cells, the first cell density adjustment was performed at 0.75 × 10 6 Cells / mL were inoculated into 30 mL of cells in a 125 mL shake flask.

[0049] The inoculated 125 mL shake flask was placed in an incubator at 37°C, 5% CO2, and 90 rpm for 24 h. Samples were taken and counted. The cell status and viability were observed to be above 95%. The second cell density adjustment was performed and the cell density was adjusted to 3.0×10 using Kangsheng Wayne293 transfection medium. 6 cells / mL, and place the cells with adjusted density into a shaker for transfection.

[0050] Prepare 30mL of recombinant protein DNA-PEI complex transfection system as follows:

[0051] Add 30 μg of recombinant protein DNA into 900 μL Kangsheng Wayne293 transfection medium to obtain DNA buffer, mix thoroughly and let stand for 5 minutes;

[0052] Add 150 μg PEI to 900 μL Kangsheng Wayne293 transfection medium to obtain PEI buffer, mix thoroughly and let stand for 5 minutes;

[0053] Add PEI buffer to DNA buffer, then add Kangsheng Wayne293 transfection medium, make the volume to 30mL, let it stand for 10 minutes to react and form recombinant protein DNA-PEI complex;

[0054] Take the cells with adjusted density out of the shaker, shake the flask gently in the same direction, slowly add the recombinant protein DNA-PEI complex, and put the cells into the shaker. Set the shaker speed to 90rpm, 105rpm, and 120rpm respectively.

[0055] The second transient transfection was performed 24h (D1) and 48h (D2) after transfection, respectively, with the following steps:

[0056] Prepare 30mL of recombinant protein DNA-PEI complex transfection system, the preparation method is the same as the first transient transfection. Take a group of cells out of the shaker at different speeds, shake the bottle gently in the same direction by hand, and slowly add the recombinant protein DNA-PEI complex. After the complex is added, put the cells into the shaker at the corresponding speed.

[0057] After 6 days of culture, or when the cell viability is less than 50%, samples are collected, counted, and the protein concentration is measured after downstream purification.

[0058] Table 3

[0059]

[0060] According to Table 3, when the cell density was 3.0×10 6 cells / mL, and the mass ratio of recombinant protein DNA to PEI was 1:5. The shaking speed was adjusted to 90rpm, 105rpm, and 120rpm. The second transient transfection was performed 24h and 48h later. It was found that the expression level of cells after the second transient transfection was higher. With the increase of the speed, the expression level also increased. When the speed was 120rpm, the expression level was the highest, and the transfection efficiency after 24h was higher than that after 48h. Subsequent additive experiments were carried out on this basis.

[0061] Example 4

[0062] Effects of the type and amount of additives added after transfection on the expression of recombinant proteins

[0063] HEK 293 cells in the logarithmic growth phase were centrifuged at 800 rpm for 5 min to resuspend the cells. Then, the cells were passaged using Kangsheng Wayne 293 transfection medium. After the third passage, that is, 24 h before transient transfection of the cells, the first cell density adjustment was performed at 0.75 × 10 6 Cells / mL were inoculated into 30 mL of cells in a 125 mL shake flask.

[0064] The inoculated 125 mL shake flask was placed in an incubator at 37°C, 5% CO2, and 120 rpm for 24 h. Samples were taken and counted. The cell status and viability were observed. The second cell density adjustment was performed when the cell density was above 95%. Kangsheng Wayne293 transfection medium was used to adjust the cell density to 3.0×10 6 cells / mL. Place the cells with adjusted density into a shaker for transfection.

[0065] Prepare 30mL of recombinant protein DNA-PEI complex transfection system as follows:

[0066] Add 30 μg of recombinant protein DNA into 900 μL Kangsheng Wayne293 transfection medium to obtain DNA buffer, mix thoroughly and let stand for 5 minutes;

[0067] Add 150 μg PEI to 900 μL Kangsheng Wayne293 transfection medium to obtain PEI buffer, mix thoroughly and let stand for 5 minutes;

[0068] Add PEI buffer to DNA buffer, then add Kangsheng Wayne293 transfection medium, make up to 30mL, and let stand for 10 minutes to allow the reaction to form a recombinant protein DNA-PEI complex.

[0069] Take the cells with adjusted density out of the shaker, shake the flask gently in the same direction by hand, slowly add the recombinant protein DNA-PEI complex, put the cells into the shaker, and set the shaker speed to 120 rpm.

[0070] The second transient transfection was performed 24 hours after the first transfection, and the preparation method of the transfection system was the same as that of the first transient transfection.

[0071] According to the DOE software Minitab, a general full factorial experiment is designed. The specific experimental plan is as follows:

[0072] Two factors: ammonium ferric citrate and tryptone;

[0073] Three levels: the concentration of ammonium ferric citrate used: 5μmol / L, 10μmol / L, 15μmol / L;

[0074] Tryptone concentration: 100g / L, 200g / L, 300g / L;

[0075] Table 4

[0076] The DOE designed experiment is as follows:

[0077]

[0078] Table 5 Corresponding concentrations of additives in DOE experiments

[0079]

[0080] Ammonium ferric citrate solutions were prepared to have final concentrations of 5 μmol / L, 10 μmol / L, and 15 μmol / L, respectively;

[0081] Prepare tryptone solutions to have final concentrations of 100 g / L, 200 g / L, and 300 g / L respectively;

[0082] 24 hours after the second transient transfection, add ammonium ferric citrate and trypsin according to the above DOE design groups;

[0083] After 6 days of culture, or when the cell viability is less than 50%, samples are collected, counted, and the protein concentration is measured after downstream purification.

[0084] Table 6

[0085]

[0086] As shown in Table 6, the expression level was the highest when ammonium ferric citrate with a final concentration of 5 μmol / L and trypsin with a final concentration of 200 g / L were added simultaneously after the second transient transfection. The transfection effect of adding ammonium ferric citrate and trypsin 24 hours after the second transient transfection was better than that of adding ammonium ferric citrate 48 hours after the second transient transfection. The higher the concentration of ammonium ferric citrate used, the less conducive it was to the increase in expression.

[0087] Example 5

[0088] Comparison of expression levels of recombinant proteins between the optimized experimental scheme and the scheme before optimization

[0089] The experimental plan before optimization is as follows:

[0090] HEK 293 cells in the logarithmic growth phase were centrifuged at 800 rpm for 5 min to resuspend the cells. Gibco Freestyle CHO medium was used for cell passage. After the third passage, that is, 24 h before transient cell transfection, the first cell density adjustment was performed at 0.5 × 10 6 Cells / mL were inoculated into 30 mL of cells in a 125 mL shake flask.

[0091] The inoculated 125 mL shake flask was placed in an incubator at 37°C, 5% CO2, and 90 rpm for 24 h. Samples were taken and counted. The cell viability was observed to be above 95%. The second cell density adjustment was performed and the cell density was adjusted to 2.0 × 10 using Gibco Freestyle CHO medium. 6cells / mL, and place the cells with adjusted density into a shaker for transfection.

[0092] Prepare 30mL of recombinant protein DNA-PEI complex transfection system as follows:

[0093] Add 30 μg of recombinant protein DNA to 900 μL Gibco Freestyle CHO medium to obtain DNA buffer, mix thoroughly and let stand for 5 min;

[0094] Add 90 μg PEI to 900 μL Gibco Freestyle CHO medium to obtain PEI buffer. Mix thoroughly and let stand for 5 minutes;

[0095] Add PEI buffer to DNA buffer, then add Gibco Freestyle CHO medium, make the volume to 30 mL, and let it stand for 10 minutes to allow the reaction to form a recombinant protein DNA-PEI complex.

[0096] Take the cells with adjusted density out of the shaker, gently shake the flask in the same direction with your hand, slowly add the recombinant protein DNA-PEI complex, put the cells into the shaker, and set the shaker speed to 90 rpm.

[0097] After 6 days of culture, or when the cell viability is less than 50%, samples are collected, counted, and the protein concentration is measured after downstream purification.

[0098] Optimize the post-transfection experiment as follows:

[0099] HEK 293 cells in the logarithmic growth phase were centrifuged at 800 rpm for 5 min to resuspend the cells. Then, the cells were passaged using Kangsheng Wayne 293 transfection medium. After the third passage, that is, 24 h before transient transfection of the cells, the first cell density adjustment was performed at 0.75 × 10 6 Cells / mL were inoculated into 30 mL of cells in a 125 mL shake flask.

[0100] The inoculated 125 mL shake flask was placed in an incubator at 37°C, 5% CO2, and 120 rpm for 24 h. Samples were taken and counted. The cell viability was observed to be above 95%. The second cell density adjustment was performed and the cell density was adjusted to 3.0×10 using Kangsheng Wayne293 transfection medium. 6 cells / mL, and place the cells with adjusted density into a shaker for transfection.

[0101] Prepare 30mL of recombinant protein DNA-PEI complex transfection system as follows:

[0102] Add 30 μg of recombinant protein DNA into 900 μL Kangsheng Wayne293 transfection medium to obtain DNA buffer, mix thoroughly and let stand for 5 minutes;

[0103] Add 150 μg PEI to 900 μL Kangsheng Wayne293 transfection medium to obtain PEI buffer, mix thoroughly and let stand for 5 minutes;

[0104] Add PEI buffer to DNA buffer, and then add to Kangsheng Wayne293 transfection medium, make up to 30mL, and let it stand for 10 minutes to allow it to react and form a recombinant protein DNA-PEI complex.

[0105] Take the cells with adjusted density out of the shaker, shake the flask gently in the same direction by hand, slowly add the recombinant protein DNA-PEI complex, put the cells into the shaker, and set the shaker speed to 120 rpm.

[0106] The second transient transfection was performed 24 hours after transfection, and the preparation method of the transfection system was the same as that of the first transient transfection.

[0107] After the cells were placed in a shaker for 24 hours, 5 μmol / L final concentration of ammonium ferric citrate and 200 g / L final concentration of trypsin were added to the cells for the second transient transfection, and the cells were placed in a shaker. After 6 days of culture, or when the cell viability was less than 50%, samples were collected, counted, and the protein concentration was measured after downstream purification.

[0108] Table 7

[0109]

[0110] From Table 7 and Figure 1 It can be seen that the expression of the optimized scheme is nearly doubled compared to the scheme before optimization.

Claims

1. A method for increasing the expression of recombinant protein in transiently transfected HEK 293 cells, characterized in that: The method comprises the following steps: after HEK 293 cells are revived and subcultured, the cells with adjusted density are added to the recombinant protein DNA-PEI complex for the first transient transfection, after the first transient transfection, the HEK 293 cells are subjected to a second transient transfection, and after the second transient transfection, additives are added to the transfection system, wherein the additives include ammonium ferric citrate and trypsin.

2. The method for increasing the expression of recombinant protein in transient transfection of HEK 293 cells according to claim 1, characterized in that: Before the first transient transfection, the cell density was adjusted to 2.5-3.5×10 6 cells / mL.

3. The method for increasing the expression of recombinant protein in transient transfection of HEK 293 cells according to claim 1, characterized in that: HEK 293 cells were transiently transfected for the second time 24h~48h after the first transient transfection.

4. The method for increasing the expression of recombinant protein in transient transfection of HEK 293 cells according to claim 1, characterized in that: Add the additives within 24 hours after the second transient transfection.

5. The method for increasing the expression of recombinant protein in transient transfection of HEK 293 cells according to claim 4, characterized in that: After adding the additives, the final concentration of ammonium ferric citrate in the culture medium is 3-8 μmol / L, and the final concentration of trypsin in the culture medium is 150-250 g / L.

6. The method for increasing the expression of recombinant protein in transient transfection of HEK 293 cells according to claim 1, characterized in that: The recombinant protein DNA-PEI complex comprises recombinant protein DNA and PEI, and the mass ratio of the recombinant protein DNA to PEI is 1:4-6.

7. The method for increasing the expression of recombinant protein in transient transfection of HEK 293 cells according to claim 1, characterized in that: During the first and second transient transfections, the shaking speed was above 120 rpm.

8. The method for increasing the expression of recombinant protein in transient transfection of HEK 293 cells according to claim 1, characterized in that: The method for adjusting the cell density before the first transient transfection includes: taking HEK 293 cells in the logarithmic growth phase for subculture, and after the third generation, adjusting the cell density for the first time according to 0.7~0.8×10 6 Cells / mL were inoculated and cultured for 20-28 h. The cell density was adjusted for the second time to 2.5-3.5×10 6 cells / mL.

9. The method for increasing the expression of recombinant protein in transient transfection of HEK 293 cells according to claim 1, characterized in that: Before subculturing HEK 293 cells, centrifuge at 700-900 rpm for 4-6 minutes to resuspend the cells.

10. The method for increasing the expression of recombinant protein in transient transfection of HEK 293 cells according to claim 8, characterized in that: After the first cell density adjustment, the cell culture conditions were: temperature of 37°C, 5% volume percent CO2, and a rotation speed of more than 120 rpm.

Images