CN111004316A

CN111004316A - Activin A and preparation method thereof

Info

Publication number: CN111004316A
Application number: CN202010002693.3A
Authority: CN
Inventors: 李绍阳; 吴佳靓; 王丁力; 游婷婷; 沈潇
Original assignee: Guangzhou Lingteng Biomedical Co ltd
Current assignee: Guangzhou Lingteng Biomedical Co ltd
Priority date: 2020-01-02
Filing date: 2020-01-02
Publication date: 2020-04-14
Anticipated expiration: 2040-01-02
Also published as: CN111004316B

Abstract

The invention relates to activin A and a preparation method thereof. The preparation method of the activin A comprises the following steps: providing a sample comprising an activin a precursor protein comprising a leader peptide and a mature peptide, the leader peptide for inhibiting activity of the mature peptide; and mixing the sample with a treatment solution and incubating to eliminate the inhibition of the leader peptide on the mature peptide to obtain the activin A, wherein the treatment solution contains lower alcohol, and the volume percentage of the lower alcohol in the mixed solution of the sample and the treatment solution is 20-60%. The activin A with high activity can be obtained by the preparation method.

Description

Activin A and preparation method thereof

Technical Field

The invention relates to the field of molecular biology, in particular to activin A and a preparation method thereof.

Background

Activins (activins), which are members of the TGF- β superfamily of cytokines, are involved in a number of biological processes, including tissue morphogenesis and repair, fibrosis, inflammation, neurodevelopment, hematopoiesis, reproductive function, and carcinogenesis.

Activin A is a homodimer of two β A subunits (hereinafter also referred to simply as "β A"), β 0A subunit can also form activin AB activin A heterodimers and activin AC activin A heterodimers with β 1B or β C subunits, respectively β A of various mammalian species has high homology.14 kDa mature human β A has 100% amino acid sequence identity with bovine β A, feline β A, mouse β A, porcine β A, and rat β A.

Activin a is involved in many important biological processes and is involved in the pathogenesis of a variety of diseases. For example, activin a can serve as a neurotrophic factor and a synaptic transmission regulator, playing important roles in various types of brain injury. In addition, activin a may be involved in the pathogenesis of various liver diseases, such as acute liver injury, chronic viral hepatitis, and certain liver malignancies. Studies have demonstrated that Activin A is involved in non-alcoholic fatty liver disease (NAFLD) (Yndested A et al, Activin A in nonalcoholic fat liver disease. vitam Horm.2011; 85: 323-42).

Of note, activin a has significant clinical utility. For example, systemic muscular atrophy treatment with activin a was successful in mice. Activin A antagonists (e.g., monoclonal antibodies) can be used to prevent muscle atrophy and death caused by tumors in certain animals. In view of the potential medicinal value of activin A in the prevention and treatment of different diseases, the preparation of activin A with high activity is of great significance. At present, a prokaryotic expression system is adopted to express activin A in some researches. Because the prokaryotic expression system lacks necessary modification, the activity of Activin A can not be better ensured, and the medicinal value of Activin A is not favorably exerted. Several studies have enabled the production of activin a modified by a near-native protein via eukaryotic expression systems. However, since the protein obtained by the eukaryotic expression system is activin a precursor protein, which contains a leader peptide and a mature peptide, the leader peptide inhibits the activity of the mature peptide, making it difficult to exert the medicinal value of activin a. Some studies have treated the activin a precursor protein with arginine, urea, or detergent chips, among others, in the hope of eliminating the inhibition of the mature peptide by the leader peptide. However, the treatment with arginine, urea, detergent chips, or the like is poor, and the activity of the obtained activin A is low, and thus cannot meet the actual demand.

Disclosure of Invention

One aspect of the present invention relates to a method for preparing activin A, comprising the steps of:

providing a sample comprising an activin a precursor protein comprising a leader peptide and a mature peptide, the leader peptide for inhibiting the activity of the mature peptide; and

and mixing and incubating the sample and a treatment solution to eliminate the inhibition of the leader peptide on the mature peptide to obtain the activin A, wherein the treatment solution contains lower alcohol, and the volume percentage of the lower alcohol in the mixed solution of the sample and the treatment solution is 20-60%.

In the preparation method of the activin A, the sample contains activin A precursor protein, the activin A precursor protein contains leader peptide and mature peptide, the leader peptide is used for inhibiting the activity of the mature peptide, the sample and the treatment solution containing lower alcohol are mixed and incubated, the volume percentage content of the lower alcohol in the mixed solution after the sample and the treatment solution are mixed is controlled to be 20-60%, the inhibition of the leader peptide on the mature peptide can be eliminated, so that the mature peptide can exert the activity, and the activin A with higher activity is obtained. The relative activity of the activin A obtained by the preparation method of the activin A is 161% through experiments.

In some embodiments, the lower alcohol is selected from at least one of ethanol, n-propanol, isopropanol, n-butanol, isobutanol, methanol.

In some embodiments, the pH of the treatment fluid is between 7.0 and 8.0.

In some embodiments, the lower alcohol is ethanol, and the volume percentage of the lower alcohol in the mixed solution after the sample is mixed with the treatment solution is 36% to 40%.

In some embodiments, the treatment fluid comprises 40% to 50% ethanol and 10mM to 30mM 4-hydroxyethylpiperazine ethanesulfonic acid by volume.

In some embodiments, the step of mixing and incubating the sample with a treatment fluid to eliminate the inhibition of the mature peptide by the leader peptide further comprises: and mixing the sample with the treatment solution, and incubating to obtain an incubation solution, and performing anion exchange chromatography to obtain the activin A.

In some embodiments, the step of subjecting the incubation solution obtained after mixing and incubating the sample with the treatment solution to anion exchange chromatography to obtain the activin a comprises:

loading the incubation solution to an equilibrated anion exchange column;

washing the loaded anion exchange column with a binding solution to bind the activin A to the anion exchange column; the binding solution comprises 10-30 mM of 4-hydroxyethyl piperazine ethanesulfonic acid and 20-60% of lower alcohol by volume percentage, and the pH value of the binding solution is 7.0-8.0; and

performing linear concentration gradient elution on the anion exchange column combined with the activin A by using a first eluent and a second eluent, and collecting an eluate of a target peak to obtain the activin A; the first eluent comprises 10-30 mM of 4-hydroxyethyl piperazine ethanesulfonic acid and 20-60% of the lower alcohol by volume percentage, and the pH value of the first eluent is 7.0-8.0; the second eluent comprises 10 mM-30 mM of 4-hydroxyethyl piperazine ethanesulfonic acid, 20-60% of the lower alcohol and 275 mM-325 mM of NaCl, and the pH of the second eluent is 7.0-8.0.

In some embodiments, the anion exchange column is a quaternary ammonium based anion exchange column;

and/or the binding solution comprises 10-30 mM of 4-hydroxyethyl piperazine ethanesulfonic acid and 36-40% of ethanol by volume percentage, and the pH value of the binding solution is 7.3-7.7;

and/or the first eluent comprises 10 mM-30 mM of 4-hydroxyethyl piperazine ethanesulfonic acid and 36% -40% of ethanol by volume percentage, and the pH of the first eluent is 7.3-7.7; the second eluent comprises 10 mM-30 mM of 4-hydroxyethyl piperazine ethanesulfonic acid, 36% -40% of ethanol and 275 mM-325 mM of NaCl, and the pH of the second eluent is 7.3-7.7.

In some embodiments, the step of mixing and incubating the sample with a treatment fluid further comprises the step of enriching the activin a precursor protein by: subjecting the sample to cation exchange chromatography to enrich the activin A precursor protein.

In some embodiments, the step of subjecting the sample to cation exchange chromatography to enrich for the activin a precursor protein comprises:

loading the sample onto the cation exchange column after equilibration;

washing the loaded cation exchange column with a binding solution to bind the activin A precursor protein to the cation exchange column, wherein the binding solution comprises 10 mM-30 mM histidine and 25 mM-75 mM NaCl, and the pH of the binding solution is 6.3-6.7; and

and washing the cation exchange column combined with the activin A precursor protein by using an eluent, and collecting an eluate of a target peak to obtain the enriched activin A precursor protein, wherein the eluent comprises 10 mM-30 mM of histidine and 275 mM-325 mM of NaCl, and the pH of the eluent is 6.3-6.7.

In one embodiment, the cation exchange column is a strong sulfopropyl cation exchange column.

Another aspect of the invention relates to activin A, which is prepared by the method for preparing activin A.

Drawings

FIG. 1 is a schematic flow chart of the process for preparing activin A according to the invention;

FIG. 2 is a plasmid map of the pMPT-activin A plasmid;

FIG. 3 is a statistical graph of cell viability of the positive recombinant cells in example 1 within 12 days of further culture after transfection for 48 h;

FIG. 4 is an electrophoretogram of an eluate containing activin A in example 2.

Detailed Description

To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

Wherein, the lower alcohol referred to herein is an alcohol having 1 to 4 carbon atoms; activin a, as used herein, refers to an activin a mature peptide.

The method for producing activin a according to an embodiment can produce activin a having a higher activity. Referring to FIG. 1, FIG. 1 is a schematic flow chart of a process for preparing activin A, in which arrows (1-1) indicate the leader peptide and arrows (1-2) indicate the mature peptide. The activin A precursor protein obtained by expressing activin A in cells includes a mature peptide and a leader peptide. The mature peptides exist mainly as dimers, and two mature peptides are linked by a disulfide bond. The preparation method of the activin A comprises the following steps of S110-S120:

s110, providing a sample, wherein the sample contains an activin A precursor protein, and the activin A precursor protein contains a phase leader peptide and a mature peptide, and the leader peptide is used for inhibiting the activity of the mature peptide.

In one embodiment, the leader peptide inhibits the activity of the mature peptide through interaction with the mature peptide. Further, the leader peptide and the mature peptide have at least one of the following interactions: hydrophobic interactions, hydrogen bonding, and van der waals forces. The interaction between the leader peptide and the mature peptide in the activin a precursor protein is not limited to the above-described interaction, and may be any other interaction that can inhibit the mature peptide.

In one embodiment, the activin A precursor protein is found in HGNC database, HGNC: 6065. Furthermore, the amino acid sequence of the leader peptide in the activin A precursor protein is shown as SEQ ID No.1, and the amino acid sequence of the mature peptide is shown as SEQ ID No. 2.

Specifically, the sequence shown as SEQ ID No.1 is: HALGGFTHRGSEPEEEEDVSQAILFPATDASCEDKSAARGLAQEAEEGLFRYMFRPSQHTRSRQVTSAQLWFHTGLDRQGTAASNSSEPLLGLLALSPGGPVAVPMSLGHAPPHWAVLHLATSALSLLTHPVLVLLLRCPLCTCSARPEATPFLVAHTRTRPPSGGERARR are provided.

The sequence shown as SEQ ID No.2 is: STPLMSWPWSPSALRLLQRPPEEPAAHANCHRVALNISFQELGWERWIVYPPSFIFHYCHGGCGLHIPPNLSLPVPGAPPTPAQPYSLLPGAQPCCAALPGTMRPLHVRTTSDGGYSFKYETVPNLLTQHCACI are provided.

In one embodiment, the sample is a culture broth of recombinant CHO cells or a culture supernatant of recombinant CHO cells.

Further, the preparation process of the sample comprises S111-S112:

s111, adding a transfection reagent, a plasmid containing an encoding gene of an activin A precursor protein and a plasmid containing a transposase gene into the CHO cell suspension, performing stable transfection, and culturing to obtain the recombinant CHO cell.

In one embodiment, the transfection reagent is polyethyleneimine. The plasmid containing the gene encoding the activin A precursor protein was the pMPT-activin A plasmid. The plasmid containing the Transposase gene is a plasmid containing a Transposase gene commonly used in the art, and may be, for example, a Super piggyBac transpose expression vector (i.e., piggyBac Transposase expression vector, cat # PB210 PA-1). The CHO cells were CHO-DG44 cells. Further, the plasmid map of the pMPT-activin A plasmid is shown in FIG. 2, in which "AmpR" is ampicillin resistance and "puroRe" is puromycin resistance in FIG. 2.

The plasmid containing the transposase gene is not limited to the above-mentioned plasmid, and may be any other plasmid containing a transposase gene commonly used in the art, and may be selected as necessary.

In one embodiment, the mass ratio of the plasmid containing the gene encoding the activin A precursor protein, the plasmid containing the transposase gene, and the transfection reagent is 0.9:0.1: 5. Further, the cell density of the CHO cell suspension was 3.0X 10⁶Individual cells/mL. The final concentration of both plasmids was 1.5. mu.g/10⁶And (4) cells. The final concentration of transfection reagent was 7.5. mu.g/10⁶And (4) cells.

In one embodiment, the CHO cell suspension further comprises serum-free medium. Further, the serum-free medium was proCHO5 medium.

In one embodiment, the transfection temperature is 37 ℃.

In one embodiment, the step of culturing the transfected CHO cells to obtain recombinant CHO cells comprises: after culturing the transfected CHO cells for 48 hours, the cell density was adjusted to 0.5X 10⁶The cells/mL were cultured by adding puromycin to a final concentration of 10. mu.g/mL, and the medium was changed every 2 days and puromycin was added to adjust the cell density to 0.5X 10⁶Individual cells/mL, and the final concentration of puromycin was made to be 10. mu.g/mL. And after the culture is finished, obtaining the recombinant CHO cell. The time from the first addition of puromycin to the end of the culture was 12 days or more.

And S112, adding sodium butyrate into the recombinant CHO cell suspension, and culturing to obtain a sample, wherein the sample contains the activin A precursor protein.

In one embodiment, the recombinant CHO cell suspension has a cell density of 3.0 x 10⁶Individual cells/mL. The concentration of sodium butyrate was 2 mmol/L. The culture temperature was 31 ℃. The cultivation time was 7 days.

Generally, the preparation of activin A by renaturation after prokaryotic expression of inclusion bodies is mainly adopted, the efficiency is low, and the activity of activin A protein cannot be ensured due to the lack of necessary modification of a prokaryotic expression system. In the embodiment, the recombinant CHO cell expressed activin A precursor protein is adopted, the glycosylation modification of the obtained activin A is closer to that of the natural activin A, and the natural structure and activity of the activin A can be ensured.

In one embodiment, the sample is a culture broth of recombinant CHO cells, and the step of S110 comprises: and (3) carrying out solid-liquid separation on the culture solution of the recombinant CHO cells, and collecting supernatant to obtain a sample. Further, the solid-liquid separation method is centrifugation. Further, the centrifugation temperature is 2 ℃ to 8 ℃. The centrifugal rotating speed is 3000 g-6000 g. The centrifugation time is 10 min-30 min.

And S120, mixing the sample with the treatment solution and incubating to eliminate the inhibition of the leader peptide on the mature peptide to obtain the activin A. The treatment liquid contains a lower alcohol. The volume percentage content of the lower alcohol in the mixed solution after the sample and the treatment solution are mixed is 20-60%.

The sample and the treatment solution containing the lower alcohol are mixed and incubated, and the volume percentage content of the lower alcohol in the mixed solution after the sample and the treatment solution containing the lower alcohol are mixed is controlled to be 20-60%, so that the inhibition of the leader peptide on the mature peptide can be eliminated, and the mature peptide can exert the activity, and the activin A with higher activity can be obtained.

In some embodiments, the lower alcohol is selected from at least one of ethanol, isopropanol, methanol. The arrangement can eliminate the interaction between the leader peptide and the mature peptide, and can avoid the influence on the structure of the mature peptide so as to obtain the activin A with higher activity.

In some embodiments, the lower alcohol is ethanol, and the volume percentage of the lower alcohol in the mixed solution after the sample is mixed with the treatment solution is 36% to 40%. Further, the treatment fluid comprises 40-50% by volume of ethanol. In some of these embodiments, the treatment fluid comprises 40%, 42%, 44%, 46%, 48%, or 50% ethanol by volume.

In some embodiments, the pH of the treatment fluid is between 7.0 and 8.0. Treatment of activin A at this pH advantageously ensures the activity of the mature peptide. Further, the pH of the treatment liquid was 7.5.

In some embodiments, the treatment fluid further comprises a buffer. Further, the buffer is 4-hydroxyethyl piperazine ethanesulfonic acid. Furthermore, the treatment solution also comprises 10 mM-30 mM of 4-hydroxyethyl piperazine ethanesulfonic acid. 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES) can better maintain the pH value of the treatment solution so as to ensure the activity of the mature peptide. Specifically, the treatment solution also comprises 15 mM-25 mM of 4-hydroxyethyl piperazine ethanesulfonic acid. More specifically, the treatment fluid also included 20mM 4-hydroxyethylpiperazine ethanesulfonic acid. In some of these embodiments, the treatment fluid further comprises 10mM, 15mM, 20mM, 25mM, or 30mM 4-hydroxyethylpiperazine ethanesulfonic acid.

The buffer is not limited to 4-hydroxyethylpiperazine ethanesulfonic acid, and may be another buffer. For example, Tris (Tris hydroxymethyl aminomethane) may be used. Furthermore, the treatment solution also comprises 10 mM-50 mM Tris.

In one embodiment, in the step of mixing and incubating the sample with the treatment solution to eliminate the inhibition of the leader peptide on the mature peptide, the mixture of the sample and the treatment solution is subjected to rotary incubation at 4 ℃. Further, the incubation time is 1.5-3 h. Further, the incubation time was 2 h.

In one embodiment, after S110 and before S120, the method further comprises the step of enriching the activin a precursor protein: the sample was subjected to cation exchange chromatography to enrich for activin a precursor protein. Samples containing higher concentrations of activin A precursor protein could be obtained by enrichment.

In one embodiment, the cation exchange chromatography process is monitored by detecting the absorbance at 280nm of the effluent of the cation exchange chromatography process during the cation exchange chromatography of the sample.

In one embodiment, the step of subjecting the sample to cation exchange chromatography to enrich for activin a precursor protein comprises S131-S134:

and S131, balancing the cation exchange column by using a balancing liquid.

In one embodiment, the balancing liquid is selected from at least one of pure water and deionized water.

In one embodiment, the cation exchange column is equilibrated with an equilibration fluid to stabilize the pH, conductivity, and absorbance at 280nm of the effluent of the equilibration process. Further, the cation exchange column is equilibrated with 5 to 10 times the column volume of the equilibration solution.

In one embodiment, the cation exchange column is a strong sulfopropyl cation exchange column, i.e., an SP column. Further, the cation exchange column was a SPSepharose Fast Flow column.

In one embodiment, the column volume of the cation exchange column is 1 mL.

And S132, loading the sample to the balanced cation exchange column.

In one embodiment, the flow rate of the sample is such that the retention time of the sample is greater than 1 min. The loading speed can ensure that the activin A precursor protein can be better bonded to the cation exchange column. Furthermore, the column volume of the cation exchange column is 1mL, and the sample loading speed is 0.2 mL/min-1 mL/min.

In some embodiments, before S132, the method further includes the following steps: diluting the sample by 1-10 times by adopting 10-30 mM histidine buffer solution, and adjusting the pH value of the diluted sample to 6-7. Further, the pH-adjusted sample was filtered through a 0.45 μm filter to remove large particulate matter. Further, the sample was diluted 1-fold with 10mM to 30mM histidine buffer, and the pH of the diluted sample was adjusted to 6.5.

And S133, washing the loaded cation exchange column by using a binding solution so as to enable the activin A precursor protein to be bound to the cation exchange column.

In some embodiments, the binding solution comprises 10mM to 30mM histidine and 25mM to 75mM NaCl. The eluent which is not bonded to the cation exchange column can be sufficiently removed by adopting the bonding solution, and the activin A precursor protein can be better bonded to the cation exchange column so as to avoid flowing through of the activin A precursor protein. Further, the binding solution comprised 20mM histidine and 50mM NaCl.

In one embodiment, the pH of the binding solution is 6.3-6.7. Further, the pH of the binding solution was 6.5.

In one embodiment, in the step of washing the loaded cation exchange column with the binding solution to bind the activin a precursor protein to the cation exchange column, the loaded cation exchange column is washed with the binding solution until the pH, conductivity, and absorbance at 280nm of the effluent of the washing process are stabilized. Further, the cation exchange is washed with a binding solution of 5 to 10 column volumes.

S134, washing the cation exchange column combined with the activin A precursor protein by using eluent, and collecting eluate of a target peak to obtain the enriched activin A precursor protein.

In one embodiment, the eluent comprises histidine in the range of 10mM to 30mM and NaCl in the range of 275mM to 325 mM. And the pH of the eluent is 6.3-6.7. The adopted eluent can more thoroughly elute the activin A precursor protein combined on the cation exchange column and can better ensure the structure and the activity of the activin A precursor protein. Further, the eluent comprises 20mM histidine and 300mM NaCl. And the pH of the eluent was 6.5.

In some embodiments, in the step of washing the cation exchange column bound with the activin a precursor protein with an eluent, the cation exchange column is washed with 2 to 5 column volumes of the eluent.

In some embodiments, in the step of collecting the eluate of the target peak, an absorbance of the effluent during elution at 280nm is detected by an ultraviolet spectrophotometer, and the eluate of the target peak is collected according to an absorbance value at 280 nm. Specifically, the collection was started at an absorbance value of 10mAu at 280nm when the target peak appeared, and stopped at an absorbance value of 20mAu at 280nm at the tail of the target peak, to obtain an eluate of the target peak.

In some embodiments, the step of collecting the eluate of the target peak is followed by validating the activin a precursor protein in the eluate of the target peak. Further, the activin a precursor protein in the eluate of the peak of interest was confirmed by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Further, the eluates that were validated to be correct were stored at 4 ℃ or-80 ℃.

In some embodiments, after the step of S134, a step of sequentially performing a regeneration treatment and a cleaning treatment on the eluted cation exchange column is further included.

In some embodiments, the step of subjecting the eluted cation exchange column to a regeneration treatment comprises: and washing the eluted cation exchange column by using a regeneration liquid and pure water in sequence. Further, the conductance of the effluent was flushed to 0 during flushing. Furthermore, the eluted cation exchange column was washed with 5 times the column volume of the regenerating solution and 5 to 10 times the pure water in this order.

In some embodiments, the regeneration fluid comprises 10mM to 30mM histidine and 1M to 2M NaCl, and the pH of the regeneration fluid is 6.3 to 6.7. Further, the regeneration liquid included 20mM histidine and 1M NaCl, and the pH of the regeneration liquid was 6.5.

In some embodiments, the step of subjecting the regenerated cation exchange column to a washing treatment comprises: the cation exchange column was washed with 1M NaOH aqueous solution, 2M NaCl aqueous solution and pure water. Further, the cation exchange column was washed with 3 column volumes of 1M NaOH aqueous solution, then with 5 column volumes of pure water, followed by 5 column volumes of 2M NaCl aqueous solution, and then with 5 to 10 column volumes of pure water until the conductivity of the effluent was 0. Further, the cation exchange column was washed with an aqueous solution of ethanol having a 20% by volume percentage and stored.

In some embodiments, the step of mixing and incubating the sample with the treatment fluid to eliminate the inhibition of the mature peptide by the leader peptide further comprises: and mixing the sample with the treatment solution, and incubating to obtain an incubation solution, and performing anion exchange chromatography to obtain the activin A.

Further, the step of subjecting the incubation solution to anion exchange chromatography to obtain activin a includes steps S141 to S144:

and S141, balancing the anion exchange column by using a balancing liquid.

In one embodiment, the anion exchange column is equilibrated with an equilibration fluid to stabilize the pH, conductivity, and absorbance at 280nm of the effluent of the equilibration process. Further, 5 to 10 times of column volume of the balancing liquid is adopted to balance the anion exchange column.

In one embodiment, the anion exchange column is a quaternary ammonium based anion exchange column, i.e., a Q column. Further, the anion exchange column was a Q Sepharose Fast Flow column.

In one embodiment, the anion exchange column has a column volume of 1 mL.

And S142, loading the incubation liquid to the balanced anion exchange column.

In one embodiment, the flow rate of the sample is such that the retention time of the sample is greater than 1 min. With this loading speed, better binding of activin a to the anion exchange column can be ensured. Furthermore, the column volume of the anion exchange column is 1mL, and the sample loading speed is 0.2 mL/min-1 mL/min.

In one embodiment, the step of loading the incubation fluid onto the equilibrated anion exchange column comprises: and (3) carrying out solid-liquid separation on the incubation liquid, collecting the supernatant, and loading the supernatant into the balanced anion exchange column. Further, the solid-liquid separation method is centrifugation. Further, the incubation solution was centrifuged at 12000rpm at 4 ℃ for 10min, and the supernatant was collected.

And S143, washing the loaded anion exchange column by using the binding solution so as to enable the activin A to be bound to the anion exchange column.

In one embodiment, the binding solution comprises 10 mM-30 mM of 4-hydroxyethyl piperazine ethanesulfonic acid and 20% -60% of lower alcohol by volume. The adoption of the binding solution can fully remove components which are not bound to the anion exchange column, and ensure that the activin A is better bound to the anion exchange column so as to avoid flowing through of the activin A. Further, the pH of the binding solution is 7.0-8.0.

Furthermore, the binding solution comprises 10 mM-30 mM of 4-hydroxyethyl piperazine ethanesulfonic acid and 36% -40% of ethanol by volume percentage, and the pH value of the binding solution is 7.3-7.7. Specifically, the binding solution comprises 20mM of 4-hydroxyethylpiperazine ethanesulfonic acid and 40% by volume of ethanol. The pH of the binding solution was 7.5.

In one embodiment, in the step of washing the loaded anion exchange column with the binding solution to bind activin A to the anion exchange column, the loaded anion exchange column is washed with the binding solution until the pH, conductivity, and absorbance at 280nm of the effluent of the washing process are stabilized. Furthermore, the anion exchange is washed by using binding solution with 5-10 times of column volume.

S144, performing linear concentration gradient elution on the anion exchange column combined with the activin A by using the first eluent and the second eluent, and collecting eluate of a target peak to obtain the activin A. The first eluent comprises 10 mM-30 mM of 4-hydroxyethyl piperazine ethanesulfonic acid and 20% -60% of lower alcohol by volume percentage, and the pH of the binding solution is 7.0-8.0. The second eluent comprises 10 mM-30 mM of 4-hydroxyethyl piperazine ethanesulfonic acid, 20-60% of lower alcohol and 275 mM-325 mM of NaCl, and the pH of the second eluent is 7.0-8.0.

Activin a was better eluted from the anion exchange column by linear concentration gradient elution.

Further, the first eluent comprises 10 mM-30 mM of 4-hydroxyethyl piperazine ethanesulfonic acid and 36% -40% of ethanol by volume percentage, and the pH value of the first eluent is 7.3-7.7; the second eluent comprises 10 mM-30 mM of 4-hydroxyethyl piperazine ethanesulfonic acid, 36-40% of ethanol and 275 mM-325 mM of NaCl, and the pH of the second eluent is 7.3-7.7. Further, the first eluent comprises 20mM 4-hydroxyethylpiperazine ethanesulfonic acid and 40% by volume ethanol, and the second eluent has a pH of 7.5. The second eluent comprises 10 mM-30 mM of 4-hydroxyethylpiperazine ethanesulfonic acid, 40% by volume of ethanol and 300mM of NaCl, and the pH of the second eluent is 7.5.

In one embodiment, in the step of performing linear concentration gradient elution on the anion exchange column bound with activin a using the first eluent and the second eluent, the linear gradient is decreased to 0% by making the mass ratio of the first eluent in the mixed solution after the first eluent and the second eluent are mixed be 100%, and the linear gradient is increased to 100% by making the mass ratio of the second eluent in the mixed solution after the first eluent and the second eluent are mixed be 0%.

In one embodiment, in the step of washing the anion exchange column to which activin a is bound with the eluent, the anion exchange column is washed with the eluent in a volume of 5 to 30 times the column volume.

In one embodiment, in the step of collecting the eluate of the target peak, an ultraviolet spectrophotometer is used to detect the absorbance of the effluent at 280nm during the elution process, and the eluate of the target peak is collected according to the absorbance value at 280 nm. Specifically, the collection was started at an absorbance value of 10mAu at 280nm when the target peak appeared, and stopped at an absorbance value of 20mAu at 280nm at the tail of the target peak, to obtain an eluate of the target peak.

In one embodiment, the step of collecting the eluate of the target peak is followed by verifying activin a in the eluate of the target peak. Further, the eluate of the peak of interest was confirmed for activin a by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis).

In one embodiment, the step of collecting the eluate of the target peak is followed by the step of preserving the eluate of the target peak. Further, the storage temperature was 4 ℃ or-80 ℃. Further, it was diluted 1-fold with HEPES of pH7.5 and 20mM, concentrated to an appropriate concentration by ultrafiltration, snap-frozen with liquid nitrogen, and stored at-80 ℃ for a long period.

In one embodiment, after the step of S143, the method further comprises the step of sequentially performing a regeneration process and a cleaning process on the eluted anion exchange column.

In one embodiment, the step of regenerating the eluted anion exchange column comprises: and washing the eluted anion exchange column by using a regeneration liquid and pure water in sequence. Further, the conductance of the effluent was flushed to 0 during flushing. Furthermore, the eluted anion exchange column was washed with 5 times the column volume of the regeneration solution and 5 to 10 times the pure water in this order.

In one embodiment, the regeneration liquid comprises 10 mM-30 mM HEPES, 36% -40% ethanol by volume and 1M-2M NaCl, and the pH value of the regeneration liquid is 7.3-7.7. Further, the regeneration liquid comprised 20mM HEPES, 40% by volume of ethanol and 1M NaCl, and the pH of the regeneration liquid was 7.5.

In one embodiment, the step of subjecting the regenerated anion exchange column to a washing treatment comprises: the anion exchange column was washed with 1M NaOH aqueous solution, 2M NaCl aqueous solution and pure water. Further, the anion exchange column was washed with 3 column volumes of 1M NaOH aqueous solution, then with 5 column volumes of pure water, then with 5 column volumes of 2M NaCl aqueous solution, and then with 5 to 10 column volumes of pure water until the conductivity of the effluent was 0. Further, the anion exchange column was washed with an aqueous solution of ethanol at 20% by volume and stored.

In the preparation method of the activin A, the sample contains activin A precursor protein, the activin A precursor protein contains leader peptide and mature peptide, the leader peptide is used for inhibiting the activity of the mature peptide, the sample and the treatment solution containing lower alcohol are mixed and incubated, the volume percentage content of the lower alcohol in the mixed solution after the sample and the treatment solution are mixed is controlled to be 20-60%, the inhibition of the leader peptide on the mature peptide can be eliminated, so that the mature peptide can exert the activity, and the activin A with higher activity is obtained. Experiments prove that the relative activity of the activin A obtained by the activin A preparation method is 97.8-99.8%.

Moreover, in the preparation method of the activin A, the sample is subjected to anion exchange chromatography, so that the activin A precursor protein in the sample can be enriched, and the large-scale preparation of the activin A is facilitated. Furthermore, by optimizing the conditions of anion exchange chromatography, the activin A precursor protein can be enriched, the structure of the activin A precursor protein cannot be changed, and the activity of the subsequently obtained activin A can be ensured.

Then, in the preparation method of the activin A, the anion exchange chromatography is performed on the incubation liquid obtained by mixing and incubating the sample and the treatment liquid, so that the activin A with high purity can be obtained, and the conditions of the anion exchange chromatography are optimized, so that the purity of the activin A can be improved, the recovery rate of the activin A is ensured, the property of the activin A is not influenced, and the activin A with good activity can be obtained.

Furthermore, in the preparation method of the activin A, the recombinant activin A precursor protein expressed by CHO cells is adopted, the glycosylation modification of the obtained activin A protein is closer to the glycosylation modification of the natural activin A protein, and the natural structure and the activity of the activin A protein can be ensured.

Finally, the preparation method of the activin A protein is simple to operate and is beneficial to large-scale preparation of the activin A.

The following is the example section.

Example 1

Preparation of recombinant CHO cells:

(1) the CHO-DG44 cells frozen in liquid nitrogen are recovered and cultured, and transfection operation can be carried out after the cell growth rate is normal and 3 passages. Cell density was adjusted to 2.0X 10 the day before transfection⁶The cell density can reach 4 multiplied by 10 per mL on the next day⁶cell/mL to 5X 10⁶Individual cells/mL. At the time of transfection, the cell density was adjusted to 3.0X 10⁶cells/mL and the medium was replaced with fresh proCHO5 medium, fresh proCHO5 medium in a volume of 5mL, to give a CHO cell suspension. Adding a transfection reagent, a plasmid containing an encoding gene of an activin A precursor protein and a plasmid containing a transposase gene into the CHO cell suspension, quickly and uniformly mixing, and then placing the mixture into a shaker at 37 ℃ for transfection. Wherein the transfection reagent is Polyethyleneimine (PEI). The plasmid containing the coding gene of the activin A precursor protein is pMPT-activin A plasmid, and the plasmid map of the plasmid is shown in figure 2, wherein the activin A precursor protein can be found in HGNC:6065 in a HGNC database, the amino acid sequence of a leader peptide in the activin A precursor protein is shown in SEQ ID No.1, and the amino acid sequence of a mature peptide is shown in SEQ ID No. 2. The plasmid containing Transposase gene is a commercially available Super piggyBac Transposase expressionon vector (i.e., piggyBac transposase expression vector, cat # PB210 PA-1). The transfection system is detailed in Table 1.

TABLE 1 CHO cell transfection System

Cell density at transfection	3.0*10⁶Individual cell/mL
		pMPT-activin A plasmid	20.25μg
Plasmid containing transposase gene	2.25μg
		PEI	112.5μg
proCHO5	5mL
		Total volume	5mL

(2) 48h after transfection, cell density was adjusted to 0.5X 10⁶cells/mL were cultured at 37 ℃ with the addition of puromycin to a final concentration of 10. mu.g/mL. The medium was changed every 2 days and puromycin was added to adjust the cell density to 0.5X 10⁶Individual cells/mL, and the final concentration of puromycin was made to be 10. mu.g/mL. After culturing for another 12 days after transfection for 48h, positive recombinant cells were obtained. The cell viability of the positive recombinant cells within another 12 days of culture after 48h of transfection is detailed in FIG. 3. FIG. 3 is a statistical graph of cell viability of positive recombinant cells within 48h of transfection and 12 days of culture.

As can be seen from FIG. 3, the cell viability of the positive recombinant cells reached more than 96% within 48h after transfection and 12 days after culture.

(3) Adjusting cell density of positive recombinant cells to 3.0 x 10⁶cells/mL, culture volume adjusted to 10 mL/tube, and added sodium butyrate to 2mM, placed at 31 ℃ for 7 days shaking culture. After the culture was completed, centrifugation was carried out at 1500rpm for 5min, and the supernatant was collected.

Example 2

First, enrichment of activin A precursor protein

1. The reagents used were:

the binding solution comprised 20mM histidine and 50mM NaCl and had a pH of 6.5.

The eluent comprises 20mM histidine and 300mM NaCl and has a pH of 6.5.

The regeneration solution contained 20mM histidine and 1M NaCl, and the pH of the regeneration solution was 6.5.

2. The experimental process comprises the following steps:

(1) preparation of a sample:

(a) the recombinant CHO cells constructed in example 1 were adjusted to 3.0X 10 in cell density⁶The cells/mL, the culture volume was adjusted to 10 mL/tube, and sodium butyrate was added to 2mM, followed by shake culture at 31 ℃ for 7 days to obtain a cell culture solution. The cell culture broth was centrifuged at 4000g for 20min at 4 ℃ and the supernatant was collected.

(b) Diluting the collected supernatant by 1 time by using a diluent, adjusting the pH of the diluted supernatant to 6.5, and filtering by using a 0.45 mu m filter membrane to obtain a sample. Histidine buffer with 20mM dilution

(2) The cation exchange column is balanced by using a balancing solution with 8 times of column volume, the Flow rate is 1mL/min, the cation exchange column is an SP Sepharose Fast Flow column, and the column volume is 1 mL. The equilibrium liquid is pure water.

(3) The sample was loaded onto the equilibrated cation exchange column at a flow rate of 1 mL/min.

(4) The cation exchange was washed with 8 column volumes of binding solution.

(5) And (3) washing the cation exchange column by using eluent with 5 times of column volume, starting collection at UV280 of more than 20mAu, and ending collection when UV280 drops to less than 20mAu to obtain eluate. The activin A precursor protein in the eluate was verified by reducing SDS-PAGE and stored at 4 ℃.

(6) The eluted cation exchange column was washed with 5 column volumes of the regeneration solution and 8 times of pure water in this order.

(7) The cation exchange column was washed with 3 column volumes of 1M NaOH aqueous solution, then with 5 column volumes of pure water, then with 3 column volumes of 2M NaCl aqueous solution, and then with 5 to 10 column volumes of pure water until the conductivity of the effluent was 0. And (3) washing and storing the cation exchange column by using an ethanol aqueous solution with the volume percentage of 20%.

Second, activation of activin A precursor protein

The eluate containing the activin a precursor protein obtained in example 1 was mixed with the treatment solution and placed at 4 ℃ to rotary-incubate the diluted sample for 2 hours to obtain an incubation solution. The volume ratio of the eluate to the treatment solution is 1: 9. the treatment fluid comprised 40% ethanol by volume, 20mM HEPES, and a pH of 7.5.

Thirdly, carrying out anion exchange chromatography on the incubation liquid

(1) The reagents used were:

the binding solution comprises 20mM HEPES and 40% by volume ethanol, and the pH of the binding solution is 7.5.

The first eluent comprised 20mM HEPES and 40% by volume ethanol, and the pH of the binding solution was 7.5.

The second eluent comprises 20mM HEPES, 40% by volume ethanol and 300mM NaCl, and the pH of the second eluent is 7.5.

The regeneration solution comprises 20mM HEPES, 40% ethanol by volume and 1M NaCl, and the pH of the regeneration solution is 7.5.

(2) Procedure of experiment

(a) The anion exchange column was equilibrated with 8 column volumes of equilibration solution. The Flow rate was 1mL/min, the anion exchange column was a Q Sepharose Fast Flow column, and the column volume was 1 mL. The equilibrium liquid is pure water.

(b) The incubation was centrifuged at 12000rpm for 10min at 4 ℃ and the supernatant was collected and applied to an equilibrated anion exchange column. The sample loading speed is 1 mL/min.

(c) The anion exchange was washed with 8 column volumes of binding solution. The flow rate was 1 mL/min.

(d) And (3) performing linear concentration gradient elution on the anion exchange column combined with the activin A by using a first eluent and a second eluent, starting collection when the UV280 is more than 20mAu, and ending collection when the UV280 is reduced to be less than 20mAu to obtain eluate containing the activin A. The elution flow rate was 1mL/min, and the linear concentration gradient elution was: the linear gradient of the first eluent with the mass ratio of 100% in the mixed liquid of the first eluent and the second eluent is reduced to 0% within 20min, and the linear gradient of the second eluent with the mass ratio of 0% in the mixed liquid of the first eluent and the second eluent is increased to 100%. The eluate was verified for activin A by SDS-PAGE and stored at 4 ℃. The results are shown in FIG. 4. In fig. 4, channel No.1 is a protein molecular weight standard; channel 2 is the band of the eluate as determined by reducing SDS-PAGE, and the band indicated by the arrow (4-1) is activin A; the lane 3 is a band of the eluate measured by non-reducing SDS-PAGE, and the band indicated by the arrow (4-2) is activin A.

(e) The eluted anion exchange column was washed with 5 column volumes of the regeneration solution and 8 times of pure water in this order.

(f) The anion exchange column was washed with 3 column volumes of 1M NaOH aqueous solution, then with 5 column volumes of pure water, then with 3 column volumes of 2M NaCl aqueous solution, and then with 8 column volumes of pure water until the conductivity of the effluent was 0. Washing and storing the anion exchange column by using an ethanol aqueous solution with the volume percentage of 20%.

As can be seen from FIG. 4, the activin A precursor protein was converted to active activin A after treatment with the treatment solution.

The prepared hormone A was tested for relative activity, which was determined as follows: reference publications the activity of activin a in the eluate containing activin a collected by anion exchange chromatography of example 2 and a commercially available activin a Protein standard, which is a rebbinant human/Mouse/Rat activin a Protein from R & D, cat # 338-AC-050, were measured for its ability to induce hemoglobin expression in K562 human chronic myelogenous leukemia cells, and the ratio of the activity of activin a Protein of example 2 to the activity of the commercially available activin a Protein standard, which is the relative activity of activin a of the present invention, was calculated; the publication is Erythroidditional biology for activity.Schwall, R.H.et al (1991). The results are shown in Table 2.

TABLE 2 relative Activity of activin A

	Relative Activity (%)
		Example 2	161

As can be seen from table 2, the preparation method of activin a of this example can obtain activin a with higher activity.

Example 3

This embodiment is generally the same as embodiment 2, except that:

1. in experiments for enrichment of activin A precursor protein, the binding solution included 10mM histidine and 25mM NaCl and the pH of the binding solution was 6.7. The eluent comprised 10mM histidine and 275mM NaCl, and had a pH of 6.7. The flow rate of the sample was 0.2 mL/min. The elution flow rate was 0.2 mL/min.

2. In the experiment of activating the activin A precursor protein, the incubation time was 1.5h, and an incubation solution was obtained. The volume ratio of the eluate to the treatment solution is 1: 9. the treatment fluid comprised 45% ethanol, 10mM HEPES, by volume, and a pH of 7.

3. In the anion exchange chromatography experiment, the binding solution comprises 10mM HEPES and 36% ethanol by volume, and the pH of the binding solution is 7.3. The first eluent comprises 10mM HEPES and 36% by volume ethanol, and the pH of the first eluent is 7.3. The second eluent comprises 10mM HEPES, 36% by volume ethanol and 275mM NaCl, and the pH of the second eluent is 7.3. The flow rate of the sample was 0.2 mL/min. The elution flow rate was 0.2 mL/min.

Example 4

This embodiment is generally the same as embodiment 2, except that:

1. in experiments for enrichment of activin A precursor protein, the binding solution included 30mM histidine and 75mM NaCl and was at pH 6.3. The eluent comprised 10mM histidine and 275mM NaCl, and had a pH of 6.3. The flow rate of the sample was 0.6 mL/min. The elution flow rate was 0.6 mL/min.

2. In the experiment of activating the activin A precursor protein, the incubation time was 2h to obtain an incubation solution. The volume ratio of the eluate to the treatment solution is 1: 9. the treatment fluid comprised 50% ethanol, 30mM HEPES by volume and had a pH of 8.

3. In the anion exchange chromatography experiment, the binding solution comprises 30mM HEPES and 38% ethanol by volume, and the pH of the binding solution is 7.7. The first eluent comprised 30mM HEPES and 38% by volume ethanol and had a pH of 7.7. The second eluent comprised 30mM HEPES, 38% by volume ethanol and 325mM NaCl, and the pH of the second eluent was 7.7. The flow rate of the sample was 0.6 mL/min. The elution flow rate was 0.6 mL/min. Eluting for 0.6min, collecting supernatant, and eluting for 0.6 min.

Example 5

This embodiment is generally the same as embodiment 2, except that:

in the experiment of activating the activin A precursor protein, the incubation time was 2h to obtain an incubation solution. The volume ratio of the eluate to the treatment solution is 1: 9. the treatment fluid comprised 40% by volume isopropyl alcohol, 20mM HEPES, and a pH of 7.5.

Example 6

This embodiment is generally the same as embodiment 2, except that:

in the experiment of activating the activin A precursor protein, the incubation time was 2h to obtain an incubation solution. The volume ratio of the eluate to the treatment solution is 1: 9. the treatment solution comprised 40% methanol by volume, 20mM HEPES, and a pH of 7.5.

Example 7

This embodiment is generally the same as embodiment 1, except that:

in the experiment of activating the activin A precursor protein, the incubation time was 2h to obtain an incubation solution. The volume ratio of the eluate to the treatment solution is 1: 9. the treatment solution comprised 40% ethanol, 20mM Tris, and pH7.5 by volume.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Sequence listing

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<120> activin A and preparation method thereof

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Claims

1. A preparation method of activin A is characterized by comprising the following steps:

2. The process for producing activin A according to claim 1, wherein the lower alcohol is at least one selected from the group consisting of ethanol, n-propanol, isopropanol, n-butanol, isobutanol, and methanol.

3. The method of producing activin A according to claim 1, wherein the pH of the treatment liquid is 7.0 to 8.0.

4. The method of claim 1, wherein the lower alcohol is ethanol, and the volume percentage of the lower alcohol in the mixture of the sample and the treatment solution is 36% to 40%.

5. The process of claim 4, wherein the treating solution comprises 40-50% by volume of ethanol and 10-30 mM of 4-hydroxyethylpiperazine ethanesulfonic acid.

6. The method of preparing activin A according to any one of claims 1-5, wherein the step of mixing and incubating the sample with a treatment solution to eliminate inhibition of the mature peptide by the leader peptide further comprises: and mixing the sample with the treatment solution, and incubating to obtain an incubation solution, and performing anion exchange chromatography to obtain the activin A.

7. The method according to claim 6, wherein the step of subjecting an incubation solution obtained by mixing and incubating the sample with the treatment solution to anion exchange chromatography to obtain the activin A comprises:

loading the incubation solution to an equilibrated anion exchange column;

8. The process for the preparation of activin A according to claim 7, wherein the anion exchange column is a quaternary ammonium-based anion exchange column;

9. The method of any one of claims 1 to 5, wherein the step of mixing and incubating the sample with a treatment solution further comprises a step of enriching the activin A precursor: subjecting the sample to cation exchange chromatography to enrich the activin A precursor protein.

10. The method of claim 9, wherein the step of subjecting the sample to cation exchange chromatography to enrich the activin a precursor protein comprises:

loading the sample onto the cation exchange column after equilibration;

11. The process for preparing activin A according to claim 10, wherein the cation exchange column is a strong sulfopropyl cation exchange column.

12. An activin A produced by the method for producing an activin A according to any one of claims 1 to 11.