CN115724929A

CN115724929A - Eukaryotic expression and purification method of ginseng TCTP protein

Info

Publication number: CN115724929A
Application number: CN202210982976.8A
Authority: CN
Inventors: 马兆成; 邓文俊
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2022-08-16
Filing date: 2022-08-16
Publication date: 2023-03-03

Abstract

The invention relates to a eukaryotic expression and purification method of ginseng TCTP protein, belonging to the technical field of protein eukaryotic expression. The invention provides a eukaryotic expression and purification method of ginseng TCTP protein, which comprises the steps of recombinant expression vector construction, pichia pastoris expression strain construction, recombinant yeast transformant and recombinant high copy strain screening, protein induction expression and purification. The method can realize the high-efficiency expression of the TCTP protein, and the obtained TCTP protein has high biological activity and low toxicity.

Description

Eukaryotic expression and purification method of ginseng TCTP protein

Technical Field

The invention relates to the technical field of protein eukaryotic expression, in particular to a method for eukaryotic expression and purification of ginseng TCTP protein.

Background

Translation Control Tumor Protein (TCTP) is a protein which is found in tumor cells of mammals at first, is highly conserved in genetic evolution, and exists not only in tumor cells but also in normal cells such as animals, plants, and yeasts. The ubiquitous presence of TCTP in organisms also accounts for the diversity and importance of its biological functions. The initial research suggests that TCTP is only a tumor-associated protein, but later researches find that TCTP not only can regulate cell processes such as cell cycle, extracellular histamine release, inflammatory reaction, stress reaction and the like, but also participates in regulating cell proliferation and differentiation, and has biological activities such as anti-apoptosis, anti-tumor and the like. TCTP is expected to be an important action target to provide a new idea for drug development.

At present, the in vitro expression of TCTP protein mostly adopts an escherichia coli prokaryotic expression system, and a eukaryotic expression system is not established.

Disclosure of Invention

The invention aims to provide a eukaryotic expression and purification method of ginseng TCTP protein. The method of the invention can realize the high-efficiency expression of the TCTP protein, and the obtained TCTP protein has high biological activity and low toxicity.

The invention provides a eukaryotic expression and purification method of ginseng TCTP protein, which comprises the following steps:

1) Connecting the ginseng TCTP protein gene fragment with a pPIC9K vector to construct a recombinant expression vector;

2) Transferring the recombinant expression vector obtained in the step 1) into a DH5 alpha competent cell for verification, and extracting a recombinant plasmid with correct verification;

3) Linearizing the recombinant plasmid obtained in the step 2), and recovering a linearized product;

4) Transferring the linearized product obtained in the step 3) into a pichia pastoris expression strain GS115 competent cell;

5) Screening His + recombinant transformants by using an MD plate to obtain recombinant yeast transformants;

6) Inoculating the recombinant yeast transformant obtained in the step 5) to a YPD plate containing G418, and screening a high-copy transformant to obtain a recombinant high-copy strain;

7) Sequentially inoculating the recombinant high-copy strain obtained in the step 6) to a BMGY culture medium and a BMMY culture medium, performing methanol induced expression, centrifuging, collecting supernatant, and filtering to obtain a recombinant protein solution;

8) Purifying the recombinant protein solution obtained in the step 7) by using a Ni-affinity chromatography column to obtain purified ginseng TCTP protein.

Preferably, the amino acid sequence of the ginseng TCTP protein is shown as SEQ ID NO.2, and the nucleotide sequence of the gene for coding the ginseng TCTP protein is shown as SEQ ID NO. 1.

Preferably, in the step 1), the nucleotide sequence of the primer for amplifying the ginseng TCTP protein gene segment is shown as SEQ ID NO.3 and SEQ ID NO. 4.

Preferably, the linearization in step 3) is performed by digestion with SacI enzyme.

The invention provides a eukaryotic expression and purification method of ginseng TCTP protein. The invention provides a complete expression and purification method of TCTP protein, which comprises cloning of TCTP gene, construction of pichia pastoris secretory expression vector, and induced expression and purification of TCTP protein. Firstly, compared with a prokaryotic expression system, the eukaryotic expression system can modify the recombinant protein to make the recombinant protein closer to natural protein, and the biological activity is high; secondly, the yeast has high growth density, can realize large-scale fermentation tank culture, has less self-secreted protein, does not produce toxic substances such as endotoxin and the like, and can obtain target protein with biological activity; the pPIC9K expression vector used by the invention contains an alpha factor signal peptide sequence, and an expression product can be secreted into a culture medium, so that the subsequent purification is simpler.

Drawings

FIG. 1 is a diagram showing the result of PCR amplification for the purification and expression of a target protein according to the present invention;

FIG. 2 is a diagram showing the PCR identification result of the PCR product and the pPIC9K vector ligation product provided by the present invention;

FIG. 3 is a diagram showing the colony PCR identification result of the recombinant yeast transformant provided by the present invention;

FIG. 4 is a diagram showing the results of colony PCR identification of high copy yeast transformants provided by the present invention;

FIG. 5 is a graph showing the results of Ni-affinity chromatography provided by the present invention;

FIG. 6 is a result chart of the protein identified and purified by the Western blot anti-His antibody provided by the invention as TCTP protein.

Detailed Description

4) Transferring the linearization product obtained in the step 3) into a pichia pastoris expression strain GS115 competent cell;

The invention connects the ginseng TCTP protein gene fragment with the pPIC9K vector to construct a recombinant expression vector. In the invention, the amino acid sequence of the ginseng TCTP protein is shown as SEQ ID NO.2 (MLVYQDLLTGDELLSDSFPYKEIENGVLWEVEGKWVVQGAVDVNIGANPSAEGGDEDEGVDDQTIKVVDIVDTFRLQEQPAFDKKQFVAYIKKYIKTLTPKLEDDKKEEFKKGIEGATKFLLGKLKDLQFFVGESMHDDGSLVFAYYKDGATDPTFLYFGHGLKEIKC), and the nucleotide sequence of the gene for coding the ginseng TCTP protein is shown as SEQ ID NO.1 (ATGTTGGTTTACCAGGATTTGTTGACCGGTGACGAGTTGTTGTCTGATTCTTTTCCATACAAGGAGATTGAAAACGGTGTTTTGTGGGAGGTTGAGGGTAAGTGGGTTGTTCAAGGTGCTGTTGATGTTAATATTGGTGCTAATCCATCTGCTGAGGGTGGTGATGAGGATGAAGGTGTTGATGATCAGACTATTAAGGTTGTTGATATTGTTGATACCTTCAGATTGCAAGAACAACCAGCTTTTGATAAGAAGCAATTTGTTGCCTATATCAAGAAATACATCAAGACATTGACCCCTAAGCTTGAAGACGATAAGAAGGAAGAGTTTAAGAAGGGTATAGAAGGTGCTACAAAATTTTTGCTGGGAAAGTTGAAGGATTTGCAATTTTTTGTTGGTGAAAGTATGCATGATGATGGTTCTTTGGTTTTTGCCTACTACAAGGATGGTGCTACTGACCCAACTTTTTTGTACTTTGGTCATGGTTTGAAGGAGATTAAGTGTCATCATCACCATCACCAT). In the present invention, the nucleotide sequence of the primer for amplifying the ginseng TCTP protein gene fragment is preferably as shown in SEQ ID NO.3 (TCTP-F: 5'-AGAGGCTGAAGCTTACGTAGAATTCATGTTGGTTTACCAGGATTTGTTG-3') and SEQ ID NO.4 (TCTP-R: 5'-GCGAATTAATTCGCGGCCGCATGGTGATGGTGATGATGACACT-3'). In the invention, the ginseng TCTP protein gene segment is preferably obtained by performing PCR amplification by using a recombinant plasmid containing the ginseng TCTP protein gene segment as a template. In the present invention, the reaction system for PCR amplification is preferably shown in table 1, and the reaction procedure for PCR amplification is preferably shown in table 2. The invention preferentially cuts the PCR product and the pPIC9K vector enzyme and then connects to obtain the recombinant expression vector. In the present invention, the cleavage is preferably performed using EcoRI and NotI restriction enzymes. In the present invention, the ligation is preferably performed using T4 DNA ligase.

After obtaining the recombinant expression vector, the invention transfers the recombinant expression vector into DH5 alpha competent cells for verification, and extracts the recombinant plasmid with correct verification. The method for transferring the gene is not particularly limited in the present invention, and a conventional transfer method known to those skilled in the art may be used. The invention preferably extracts the recombinant plasmid which is verified to be correct, and the method for extracting the plasmid is not particularly limited, and the plasmid extraction method which is well known to the technicians in the field can be adopted for extraction.

After the recombinant plasmid is obtained, the invention linearizes the recombinant plasmid and recovers a linearized product. In the present invention, the linearization is preferably performed by digestion with SacI enzyme.

After the linearized product is obtained, the linearized product is transferred into a pichia pastoris expression strain GS115 competent cell. In the present invention, the transfer method preferably includes a shock conversion method, the condition of the shock conversion method is preferably 1500V, and the shock time is 4 to 10ms. After electric shock, the method preferably immediately adds precooled sorbitol solution to dilute the bacterial liquid, and then stands for culture.

After the linearized product is transferred into a pichia pastoris expression strain GS115 competent cell, the invention adopts an MD plate to screen His + recombinant transformants to obtain the recombinant yeast transformants. In the invention, preferably, after static culture, the bacterial liquid is coated on an MD culture medium for inverted culture. After inverted culture, the invention preferably identifies whether the monoclonal is a positive strain by bacterial liquid PCR. In the present invention, a band is shown, and it is judged to be positive if it is 651bp in size (nucleic acid fragment size + cleavage site to 3' AOX).

After obtaining the recombinant yeast transformant, the invention inoculates the recombinant yeast transformant to a YPD plate containing G418, and screens a high-copy transformant to obtain the recombinant high-copy strain.

In the present invention, the recombinant yeast transformants are preferably inoculated into YPD plates containing G418 at different concentrations, respectively, and high-copy transformants are selected to obtain recombinant high-copy strains. In the present invention, the concentration of G418 is preferably 0,0.5,1,2,3 and 4mg/ml.

In the practice of the present invention, transformants screened on YPD plates with a G418 concentration of 3mg/ml are preferably used as high copy strains.

After obtaining the high copy strain, sequentially inoculating the recombinant high copy strain to a BMGY culture medium and a BMMY culture medium, performing methanol induced expression, centrifuging, collecting supernatant, and filtering to obtain a recombinant protein solution. In the invention, the BMGY medium takes distilled water as a solvent and contains the following components in concentration of 1L: 20g tryptone, 10g yeast extract, 100ml 10% glycerol, 100ml 1M sodium phosphate buffer, 100ml 10 times yeast nitrogen source basal medium (YNB) and 2ml 500 times biotin; the BMGY contains glycerol and is used for expressing the amplification of the pichia pastoris recombinant strain. In the invention, the BMMY culture medium takes distilled water as a solvent and contains the following components in concentration by 1L: 20g tryptone, 10g yeast extract, 100ml of 1M sodium phosphate buffer, 100ml of 10-fold YNB and 2ml of 500-fold biotin; the BMMY contains methanol for subsequent secretory expression of the induced protein. In the present invention, the methanol is preferably supplemented every 24 hours, and the final concentration of methanol is preferably 1%. In the present invention, the induction is preferably stopped at a time when the expression amount of the protein is maximized; in the specific implementation process of the invention, the expression amount of protein is determined by SDS-PAGE detection of every 24h of collected bacterial liquid, and the induction time is preferably 96h.

After the recombinant protein solution is obtained, the recombinant protein solution is purified by a Ni-affinity chromatography column to obtain purified ginseng TCTP protein. In the invention, the preferred recombinant protein solution is mixed with NTA-Ni filler and then incubated, and then put into a chromatographic column after incubation. In the present invention, it is preferable that the washing is performed using a washing solution (Wash buffer) and then the Elution is performed using an eluent (Elution buffer). In the present invention, it is particularly preferable to Wash 2 to 5 column volumes with Wash buffer (50 mM Tris-HCl (pH 7.4), 150mM NaCl, and 10mM Imidazole) and collect the flow-through solution for detecting the elution of non-specific hetero-proteins; then, eluting with an Elution buffer (50 mM Tris-HCl (pH7.4), 150mM NaCl) containing 100mM and 200mM imidazole in sequence, and collecting each eluted product for detecting the Elution condition of the target protein; finally, the protein was strongly eluted with 500mM imidazole buffer (50 mM Tris-HCl (pH 7.4), 150mM NaCl) and the strongly bound protein was washed off.

The following embodiments further describe the eukaryotic expression and purification method of TCTP protein of ginseng according to the present invention in detail, and the technical solution of the present invention includes, but is not limited to, the following embodiments.

Example 1

The specific steps of TCTP protein expression and purification are as follows:

1. molecular cloning and vector construction: PCR amplification of TCTP is carried out by taking pPIC9-TCTP recombinant plasmid synthesized by Beijing engine as template to obtain PCR product (shown in figure 1, M and 1,2 in figure 1 respectively represent DNA Marker, amplified

bands

1 and 2. Product size 539 bp). Designing a primer according to the nucleic acid sequence of TCTP amino acid sequence conversion published by Uniprot, wherein the forward primer comprises the following components: 5'-AGAGGCTGAAGCTTACGTAGAATTCATGTTGGTTTACCAGGATTTGTTG-3' (SEQ ID NO. 5); reverse primer: 5'-GCGAATTAATTCGCGGCCGCATGGTGATGGTGATGATGACACT-3' (SEQ ID NO. 6); and adding 16-25 base overlap sequences of forward or reverse direction of the vector containing EcoRI/NotI enzyme cutting sites at two ends of the primer respectively. The expression vector pPIC9K was double-digested with EcoRI and NotI restriction enzymes, and the PCR product and the digested vector pPIC9K (purchased from Novagen) were ligated using T4 DNA to construct a recombinant expression vector. Transferring the recombinant expression vector into escherichia coli DH5 alpha competence, screening on an LB solid plate containing ampicillin with the final concentration of 100mg/ml, and identifying the positive clones through colony PCR (as shown in figure 2, figure 2 is that PCR products and pPIC9K vectors are connected and transformed into the escherichia coli competence, then screening agarose gel images of positive clones through the LB solid plate, wherein M is DL2000, 100-2000bp, 1-5 are respectively strips after 5 single clones selected from the LB solid plate are run with glue, the size is 912bp (the size of 5' AOX1-EcoR I plus the size of fragments), sequencing and identifying, carrying out plasmid extraction on correct recombinant plasmids verified by sequencing, and storing at-20 ℃ for later use.

TABLE 1 PCR reaction System (50. Mu.l)

TABLE 2 PCR reaction procedure

TABLE 3 connection System

2. Obtaining of recombinant yeast transformant:

the recombinant plasmid is linearized by Sac I enzyme and transferred into the pichia pastoris GS115 competence prepared in advance by an electric shock transformation method. Setting parameters: the voltage is 1500V, the electric shock time is 4-10 ms, and the electric revolving cup is taken out after the electric shock is finished; immediately adding 1ml of precooled sorbitol solution (1 mol/L) diluted bacteria liquid on a super clean bench, sucking, uniformly mixing, transferring to a 1.5ml sterile centrifuge tube, and standing and culturing for 1h at 30 ℃;4000r/min, centrifuging for 1min, and discarding part of supernatant. After leaving about 100. Mu.l of supernatant, sucking and mixing the supernatant, evenly spreading the supernatant on an MD culture medium, carrying out inverted culture at 30 ℃ for 2-3 d, and identifying whether the monoclonal is a positive strain or not by bacterial liquid PCR (as shown in figure 3, M is DL2000,1-16 are 16 picked monoclonal antibodies, M is a 3 rd strip corresponding to 1000bp, and a target strip is near the strip with the size of 903 bp).

3. Obtaining of high-copy transformants:

the bacterial liquid of the recombinant yeast transformant selected on the MD plate is diluted by 10 times, and then the bacterial liquid is spotted on YPD plates containing G418 with different concentrations (0 mg/ml, 0.25mg/ml, 0.5mg/ml, 1mg/ml,2mg/ml and 4 mg/ml), and the bacterial liquid is cultured for 2-3 days at 30 ℃ to select the recombinant transformant containing multiple copies of TCTP gene insertion. Recombinant high-copy strains screened on G418-YPD plates and colonies transformed with pPIC9K in no-load were picked up in YPD liquid medium and cultured at 30 ℃. Positive was identified by PCR of bacterial suspension (as shown in FIG. 4, where the band size was 907bp (5' AOX1 vector primer + fragment size 539 bp). Amplification was somewhat band and positive in size).

4. Protein expression and purification:

respectively inoculating the successfully identified GS115-pPIC9K-TCTP-His (GS 115 is a pichia pastoris strain, pPIC9K is an expression vector, TCTP is a protein abbreviation name, and His is a label on the vector) and the strains of the pPIC9K vector into 2 culture bottles to a 250ml conical flask filled with 50ml of BMGY culture medium (5 ml of YNB and 100 mul of biotin are added into 45ml of BMGY), culturing for 1-2 d at 30 ℃ to ensure that the OD (OD) of a bacterial solution is ₆₀₀ >2. Transferring the BMGY bacterial liquid into a 500ml sterilized centrifugal bottle, centrifuging at 3300r/min at room temperature for 10min, removing supernatant, and collecting thalli. The cells were resuspended in BMMY medium (225 ml BMMY with 25ml YNB and 500. Mu.l biotin) to OD ₆₀₀ =1.0, and placed in a 1L Erlenmeyer flask for induction expression. Methanol is supplemented every 24h for induction to make the final concentration of methanol be 1% until the optimal induction time is reached. After 0h, 24h, 48h, 72h and 96h of induction, 1ml of induction bacterial liquid is taken for detecting whether the protein is expressed or not. Centrifuging the bacterial liquid at 12000 Xg for 5min, collecting supernatant and precipitate, and storing at-40 deg.C for detection. Carrying out mass expression on strains which are verified to be correct, centrifuging for 30min at 4000 Xg and 4 ℃, and taking supernatant; filter sterilized with a 0.22 μm filter. FiltrationThe bacterial liquid and NTA-Ni filler are incubated for 30min at the temperature of 4 ℃ by shaking at a low speed in a shaking table; filling the incubated bacteria liquid and the mixed liquid of NTA-Ni filler into a chromatographic column, and naturally settling to ensure that no bubble exists in the filler; washing 2-5 column volumes with Wash buffer (50 mM Tris-HCl (pH7.4), 150mM NaCl,10mM Imidazole), and collecting the flow-through solution (W) for detecting the elution of non-specific hybrid protein; then, eluting with an Elution buffer (50 mM Tris-HCl (pH7.4), 150mM NaCl) containing 100mM and 200mM imidazole in sequence, and collecting each of the eluates (E1, E2) for detecting the Elution condition of the target protein; finally, strong elution is carried out by using 500mM imidazole buffer solution (50 mM Tris-HCl (pH7.4), 150mM NaCl), and the protein (E3) with strong binding force is washed; the molecular weight and purity of the recombinant protein were determined by 12% SDS-PAGE (FIG. 5, E1 shows a band indicating that the 100mM elution buffer can elute most of the desired protein, E2 also shows a band indicating that the eluted protein concentration is not high and the band is shallow; E3 shows no band indicating that the 200mM elution buffer can elute all of the desired protein, resulting in no protein in the solution eluted with 500mM imidazole). The purified protein can be directly used for biochemical experiments or frozen by liquid nitrogen and stored at-80 ℃. The purified TCTP recombinant protein was verified by WB using anti-His antibody (band size between 20-25kD, target protein size about 20kD, and figure 5 size are identical), and binding of the target protein with the antibody hybridized the target protein, indicating successful expression and purification of the target protein.

The invention establishes a eukaryotic expression system for TCTP protein exogenous expression. Compared with a prokaryotic expression system, the protein expressed by the yeast secretes less foreign protein, and is more suitable for subsequent purification; no inclusion body is produced, and the biological activity is higher; yeast secretes fewer toxic substances.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. A eukaryotic expression and purification method of ginseng TCTP protein comprises the following steps:

2. The method according to claim 1, wherein the amino acid sequence of the ginseng TCTP protein is represented by SEQ ID No.2, and the nucleotide sequence of the gene encoding the ginseng TCTP protein is represented by SEQ ID No. 1.

3. The method according to claim 1, wherein in the step 1), the nucleotide sequence of the primer for amplifying the ginseng TCTP protein gene fragment is shown as SEQ ID No.3 and SEQ ID No. 4.

4. The method of claim 1, wherein the linearization of step 3) is performed using a SacI enzyme.