Purification method of bovine serum albumin

Abstract

The invention discloses a purification method of bovine serum albumin. The purification method comprises the following steps: S1, pre-treating bovine plasma to remove impurities and fibrin; s2, adsorption with tourmaline or sepiolite; s3, protein precipitation through heat shock; and S4, carrying out heparin-agarose chromatography. The bovine serum albumin sample is purified by combining tourmaline or sepiolite with heparin-agarose chromatography, nuclease in the bovine serum albumin sample can be removed, and the product purity is improved; and one-step heat shock is combined with heparin-agarose chromatography, so that the energy consumption is reduced, reagents such as ethanol influencing the protein activity are prevented from being used, the recovery yield is high, and large-scale production is facilitated.

Description

Technical Field

[0001] The invention relates to a method for purifying bovine serum albumin, and belongs to the technical field of albumin purification. Background Art

[0002] Bovine serum albumin (BSA) is the largest protein drug produced and used to date. The bovine serum albumin market is valued at $5.58 billion in 2021 and is expected to reach $18.42 billion by 2029, with a compound annual growth rate of 16.1% during the forecast period of 2022-2029. It is mainly used in treatment, drug delivery, culture medium, vaccine components and other fields. At present, the market demand for BSA is very large, with an annual demand of 600 tons in the international market and 70 tons in my country, and will continue to increase with the improvement of living standards and medical conditions.

[0003] However, the existing bovine serum albumin purification schemes include ammonium sulfate salting out method and cold ethanol precipitation method. However, the ammonium sulfate salting out method has the disadvantages of strong corrosiveness of ammonium sulfate and complicated production process. The ethanol used in the cold ethanol precipitation method is a non-specific precipitant. Although it has a significant effect on BSA production, the product purity is not very high; ethanol as an organic solvent may also cause protein aggregation or denaturation. Summary of the invention

[0004] The object of the present invention is to provide a method for purifying bovine serum albumin, which can effectively improve the purity and yield of antibodies.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is:

[0006] A method for purifying bovine serum albumin comprises the following steps:

[0007] S1, bovine plasma pretreatment to remove impurities and fibrin;

[0008] S2, tourmaline or sepiolite adsorption;

[0009] S3, heat shock precipitated protein;

[0010] S4, heparin-agarose chromatography.

[0011] Preferably, in step S1, the specific method for removing impurities and fibrin from bovine plasma is:

[0012] The frozen bovine plasma was melted in running water at 15-30°C and then filtered to remove impurities; then the two were mixed in a ratio of 1 ml plasma / 5-20 ul 30% CaCl2 solution, stirred overnight, and sieved to remove fibrin.

[0013] Preferably, 200-500 mesh silk cloth is used for filtering, and 5-30 mesh screen is used for sieving.

[0014] Preferably, in step S2, the specific method of adsorption of tourmaline or sepiolite is:

[0015] Tourmaline or sepiolite is dispersed in a buffer solution to prepare an adsorbent dispersion, and a bovine serum albumin sample obtained by pre-treatment to remove impurities and fibrin is dispersed in a buffer solution to prepare a bovine serum dispersion. The adsorbent dispersion is then added to the bovine serum dispersion, stirred, and centrifuged to collect the supernatant.

[0016] Preferably, the buffer is NaAc buffer with a concentration of 10-30 mM and a pH of 7-8.

[0017] Preferably, in the adsorbent dispersion, the concentration of tourmaline or sepiolite is (3-8) g / 100 ml;

[0018] In the bovine serum dispersion, the concentration of the bovine serum albumin sample is (0.5-1) g / ml;

[0019] Moreover, the mixing volume ratio of the adsorbent dispersion liquid to the bovine serum dispersion liquid is (3-8):1.

[0020] Preferably, in step S3, the specific method of heat shock protein precipitation is:

[0021] Heating: Filter the supernatant obtained after adsorption of tourmaline or sepiolite, add water to the filtrate and control the protein concentration to 50±5 mg / ml to obtain an albumin solution; add sodium octanoate to the albumin solution under stirring, adjust the pH to 4-6 with acid solution, heat to 60-70°C, and maintain for 2-4 hours;

[0022] Cooling and filtering: Under stirring, cool the heated albumin solution to 20-30°C with a refrigerant, collect the clear liquid by filtration, adjust the pH of the clear liquid to 7.4±0.2 with an alkali solution, and filter again;

[0023] Desalting: The filtered clear liquid is concentrated and replaced with a buffer solution, then filtered and dialyzed to obtain a protein precipitate.

[0024] Preferably, the amount of sodium caprylate added is 3-5‰ of the mass of the albumin solution;

[0025] The acid solution is 0.5-2M hydrochloric acid;

[0026] The alkali solution is 0.1-0.5M NaOH;

[0027] The buffer is NaAc buffer with a concentration of 10-30 mM and a pH of 4-6.

[0028] Preferably, the specific method of heparin-agarose chromatography is:

[0029] Filler activation and equilibration: Heparin-agarose filler is loaded into the chromatography column, and activated and equilibrated with activation solution and buffer solution in sequence;

[0030] Loading, washing and dissociation: Take the protein precipitate obtained after heat shock precipitation of protein for loading, then use buffer to wash, and then use dissociation solution for dissociation, collect the eluted components, desalt, concentrate and dialyze.

[0031] Preferably, the activation solution is 0.05-0.2M NaOH; the buffer is NaAc buffer with a concentration of 10-30mM and a pH of 4-6; and the dissociation solution is a mixture of NaAc buffer with a concentration of 10-30mM and a pH of 4-6 and NaCl buffer with a concentration of 1-2M and a pH of 4-6.

[0032] The beneficial effects of the present invention are:

[0033] 1. Purification of bovine serum albumin samples by using tourmaline or sepiolite combined with heparin-agarose chromatography can remove nucleases in bovine serum albumin samples and improve product purity;

[0034] 2. One-step heat shock combined with heparin-agarose chromatography reduces energy consumption and avoids the use of reagents such as ethanol that affect protein activity. It has a high recovery rate and is easy to scale up. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The SDS-Page electrophoresis pattern in Example 1; wherein: 1 is the standard protein maker, 2 is the protein band after heat shock, and 3 is the protein band after heparin-agarose purification;

[0036] Figure 2 It is the SDS-Page electrophoresis pattern in Example 2; wherein: 1 is the standard protein maker, 2 is the protein band after heat shock, and 3 is the protein band after heparin-agarose purification. DETAILED DESCRIPTION

[0037] Example 1

[0038] This embodiment provides a method for purifying bovine serum albumin, comprising the following steps:

[0039] S1. Pretreatment of bovine plasma to remove impurities and fibrin:

[0040] 5 g of frozen bovine plasma was melted in running water at 20-25°C, and then filtered through silk cloth (400 mesh) to remove impurities; then the two were mixed in a ratio of 1 ml plasma / 10 ul 30% CaCl2 solution, stirred overnight, and squeezed and filtered through a 10-20 mesh sieve to remove fibrin to obtain a bovine serum albumin sample.

[0041] S2, Tourmaline adsorption:

[0042] The sterilized tourmaline was dispersed in 20mM NaAc buffer (pH7.5) at a ratio of 5g / 100ml to prepare an adsorbent dispersion, and the bovine serum albumin sample was dispersed in 20mM NaAc buffer (pH7.5) at a ratio of 0.5g / ml to prepare a bovine serum dispersion. The adsorbent dispersion was then added to the bovine serum dispersion at a mixing volume ratio of 5:1, stirred, and then centrifuged to collect the supernatant.

[0043] S3, heat shock precipitated protein:

[0044] Heating: Filter the supernatant with a 0.45 μm membrane filter, then add water to the filtrate and control the protein concentration at 50 ± 5 mg / ml to obtain an albumin solution; under stirring, add sodium octanoate (4‰ of the mass of the albumin solution) to the albumin solution, and use 1 M hydrochloric acid to adjust the pH to 6.0, heat to 68 ° C, and maintain for 3 hours.

[0045] Cooling and filtration: transfer the above materials to a refrigerant tank (refrigerant 2-8°C), cool them down to 25°C under stirring, filter the cooled materials using a hollow drum centrifuge, collect the clear liquid, adjust the pH of the clear liquid to 7.4±0.2 with 0.1M NaOH, and filter with a 0.45μm membrane filter.

[0046] Desalting: The filtered clear liquid was fed into a hollow fiber membrane system, the sample was concentrated to the same volume as the raw material, and then replaced with 6 times the volume of 20mM NaAc buffer (pH5.2). After replacement, it was filtered through a 0.45μm membrane filter and dialyzed to obtain a protein precipitate.

[0047] S4, Heparin-agarose chromatography:

[0048] Filler activation and equilibration: Heparin-agarose filler was loaded into a plexiglass chromatography column. First, 5-10 column volumes of 0.1 M NaOH were used to activate the filler. Then, 5 column volumes of 20 mM NaAc (sodium acetate, pH 5.2) buffer were used to equilibrate the column. After equilibration, the protein detector was adjusted to zero and the sample was prepared.

[0049] Loading, washing and dissociation: Take 2 times the volume of the chromatographic column for loading, and then use 5-10 times the column volume of 20mM NaAc (sodium acetate, pH5.2) buffer to wash until the protein detector reads "0"; then use 5-10 times the column volume of 20mM NaAc buffer (pH5.2) + 1.4M NaCl buffer (pH5.2) for dissociation, collect samples when the protein detector reads higher than 0.2, and stop collecting samples when it is lower than 0.2. Collect the eluted components, desalt and concentrate, dialyze and freeze-dry.

[0050] SDS-Page electrophoresis pattern Figure 1 As shown. After testing, the purity of the obtained BSA was 99% and the yield was 52%.

[0051] Example 2

[0052] This embodiment provides a method for purifying bovine serum albumin, comprising the following steps:

[0053] S1. Pretreatment of bovine plasma to remove impurities and fibrin:

[0054] 5 g of frozen bovine plasma was melted in running water at 20-25°C, and then filtered through silk cloth (400 mesh) to remove impurities; then the two were mixed in a ratio of 1 ml plasma / 10 ul 30% CaCl2 solution, stirred overnight, and squeezed and filtered through a 10-20 mesh sieve to remove fibrin to obtain a bovine serum albumin sample.

[0055] S2. Sepiolite adsorption:

[0056] The sterilized sepiolite was dispersed in 20mM NaAc buffer (pH7.5) at a ratio of 5g / 100ml to prepare an adsorbent dispersion, and the bovine serum albumin sample was dispersed in 20mM NaAc buffer (pH7.5) at a ratio of 0.5g / ml to prepare a bovine serum dispersion. The adsorbent dispersion was then added to the bovine serum dispersion at a mixing volume ratio of 5:1, stirred, and then centrifuged to collect the supernatant.

[0057] S3, heat shock precipitated protein:

[0058] Heating: Filter the supernatant with a 0.45 μm membrane filter, then add water to the filtrate and control the protein concentration at 50 ± 5 mg / ml to obtain an albumin solution; under stirring, add sodium octanoate (4‰ of the mass of the albumin solution) to the albumin solution, and use 1 M hydrochloric acid to adjust the pH to 6.0, heat to 68 ° C, and maintain for 3 hours.

[0059] Cooling and filtration: transfer the above materials to a refrigerant tank (refrigerant 2-8°C), cool them down to 25°C under stirring, filter the cooled materials using a hollow drum centrifuge, collect the clear liquid, adjust the pH of the clear liquid to 7.4±0.2 with 0.1M NaOH, and filter with a 0.45μm membrane filter.

[0060] Desalting: The filtered clear liquid was fed into a hollow fiber membrane system, the sample was concentrated to the same volume as the raw material, and then replaced with 6 times the volume of 20mM NaAc buffer (pH5.2). After replacement, it was filtered through a 0.45μm membrane filter and dialyzed to obtain a protein precipitate.

[0061] S4, Heparin-agarose chromatography:

[0062] Filler activation and equilibration: Heparin-agarose filler was loaded into a plexiglass chromatography column. First, 5-10 column volumes of 0.1 M NaOH were used to activate the filler. Then, 5 column volumes of 20 mM NaAc (sodium acetate, pH 5.2) buffer were used to equilibrate the column. After equilibration, the protein detector was adjusted to zero and the sample was prepared.

[0063] Loading, washing and dissociation: Take 2 times the volume of the chromatographic column for loading, and then use 5-10 times the column volume of 20mM NaAc (sodium acetate, pH5.2) buffer to wash until the protein detector reads "0"; then use 5-10 times the column volume of 20mM NaAc buffer (pH5.2) + 1.4M NaCl buffer (pH5.2) for dissociation, collect samples when the protein detector reads higher than 0.2, and stop collecting samples when it is lower than 0.2. Collect the eluted components, desalt and concentrate, dialyze and freeze-dry.

[0064] SDS-Page electrophoresis pattern Figure 2 As shown. After testing, the purity of the obtained BSA was 99% and the yield was 53%.

[0065] Comparative Example 1

[0066] The method is basically the same as Example 1, except that the tourmaline adsorption step is not performed in Comparative Example 1.

[0067] Comparative Example 2

[0068] The method is substantially the same as Example 1, except that the heparin-agarose chromatography step is not performed in Comparative Example 2.

[0069] Comparative Example 3

[0070] It is basically the same as Example 2, except that in Comparative Example 3, the step of sepiolite adsorption is not performed.

[0071] Comparative Example 4

[0072] The method is substantially the same as Example 2, except that the heparin-agarose chromatography step is not performed in Comparative Example 4.

[0073] The effect of removing nuclease by the purified bovine serum albumin in Examples 1-2 and Comparative Examples 1-4 was detected (for the detection method, see the relevant content disclosed in the patent with publication number CN 108864278 B). The average Ct values ​​of different BSA samples amplified in the DNA and RNA systems are shown in Tables 1 and 2, respectively.

[0074] Table 1 Average Ct values ​​of different BSA samples amplified in DNA system

[0075] BSA Sample Ct value Untreated BSA 33.27 Example 1 31.57 Example 2 31.46 Comparative Example 1 33.35 Comparative Example 2 33.47 Comparative Example 3 33.32 Comparative Example 4 33.43

[0076] Table 2 Average Ct values ​​of different BSA samples amplified in RNA system

[0077] BSA Sample Ct value Untreated BSA 32.31 Example 1 23.28 Example 2 23.17 Comparative Example 1 24.45 Comparative Example 2 24.52 Comparative Example 3 24.37 Comparative Example 4 24.42

[0078] It can be seen from Table 1 and Table 2 that in the two systems, the Ct values ​​of the purified BSA samples in Example 1 and Example 2 are somewhat advanced relative to the untreated BSA samples and the BSA samples in Comparative Examples 1-4, indicating that the method provided by the present invention is effective in removing nucleases.

[0079] The above is only a preferred implementation of the patent of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the patent of the present invention. These improvements and modifications should also be regarded as the scope of protection of the patent of the present invention.

Claims

1. A method for purifying bovine serum albumin, characterized in that: The steps include: S1, pretreatment of bovine plasma to remove impurities and fibrin; S2, tourmaline or sepiolite adsorption; S3, heat shock precipitated protein; S4, heparin-agarose chromatography.

2. The method for purifying bovine serum albumin according to claim 1, characterized in that: In step S1, the specific method for removing impurities and fibrin from bovine plasma is: The frozen bovine plasma was melted in running water at 15-30°C and then filtered to remove impurities; then the two were mixed in a ratio of 1 ml plasma / 5-20 ul 30% CaCl2 solution, stirred overnight, and sieved to remove fibrin.

3. The method for purifying bovine serum albumin according to claim 2, characterized in that: Use 200-500 mesh silk cloth for filtering and 5-30 mesh screen for sieving.

4. The method for purifying bovine serum albumin according to claim 1, characterized in that: In step S2, the specific method of adsorption of tourmaline or sepiolite is: Tourmaline or sepiolite is dispersed in a buffer solution to prepare an adsorbent dispersion, and a bovine serum albumin sample obtained by pre-treatment to remove impurities and fibrin is dispersed in a buffer solution to prepare a bovine serum dispersion. The adsorbent dispersion is then added to the bovine serum dispersion, stirred, and centrifuged to collect the supernatant.

5. The method for purifying bovine serum albumin according to claim 4, characterized in that: The buffer is NaAc buffer with a concentration of 10-30 mM and a pH of 7-8.

6. The method for purifying bovine serum albumin according to claim 4, characterized in that: In the adsorbent dispersion, the concentration of tourmaline or sepiolite is (3-8) g / 100 ml; In the bovine serum dispersion, the concentration of the bovine serum albumin sample is (0.5-1) g / ml; Moreover, the mixing volume ratio of the adsorbent dispersion liquid to the bovine serum dispersion liquid is (3-8):

1.

7. The method for purifying bovine serum albumin according to claim 1, characterized in that: In step S3, the specific method of heat shock protein precipitation is: Heating: Filter the supernatant obtained after adsorption of tourmaline or sepiolite, add water to the filtrate and control the protein concentration to 50±5 mg / ml to obtain an albumin solution; add sodium octanoate to the albumin solution under stirring, adjust the pH to 4-6 with acid solution, heat to 60-70°C, and maintain for 2-4 hours; Cooling and filtering: Under stirring, cool the heated albumin solution to 20-30°C with a refrigerant, collect the clear liquid by filtration, adjust the pH of the clear liquid to 7.4±0.2 with an alkali solution, and filter again; Desalting: The filtered clear liquid is concentrated and replaced with a buffer solution, then filtered and dialyzed to obtain a protein precipitate.

8. The method for purifying bovine serum albumin according to claim 7, characterized in that: The amount of sodium octanoate added is 3-5‰ of the mass of the albumin solution; The acid solution is 0.5-2M hydrochloric acid; The alkali solution is 0.1-0.5M NaOH; The buffer is NaAc buffer with a concentration of 10-30 mM and a pH of 4-6.

9. The method for purifying bovine serum albumin according to claim 1, characterized in that: The specific method of heparin-agarose chromatography is: Filler activation and equilibration: Heparin-agarose filler is loaded into the chromatography column, and activated and equilibrated with activation solution and buffer solution in sequence; Loading, washing and dissociation: Load the protein precipitate obtained after heat shock protein precipitation, wash it with buffer, and then dissociate it with dissociation solution. Collect the eluted components, desalt, concentrate and dialyze them.

10. The method for purifying bovine serum albumin according to claim 9, characterized in that: The activation solution is 0.05-0.2M NaOH; the buffer is NaAc buffer with a concentration of 10-30 mM and a pH of 4-6; the dissociation solution is a mixture of NaAc buffer with a concentration of 10-30 mM and a pH of 4-6 and NaCl buffer with a concentration of 1-2 M and a pH of 4-6.

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