CN114958889A - Preparation method of high-activity FGF7 protein - Google Patents

Abstract

The invention discloses a preparation method of FGF7 protein with high activity. The preparation method comprises the following steps: 1) constructing pRSF-SUMO-rmFGF7 recombinant plasmid; 2) transforming the recombinant plasmid into host bacteria, inducing expression, and collecting bacteria; 3) carrying out ultrasonic crushing and centrifugation on the thalli, and collecting supernatant; 4) eluting the supernatant through a nickel column, and collecting the eluent to obtain a primary purified substance; 5) adding SUMO protease into the primary purified product, performing dialysis enzyme digestion, eluting the enzyme-cleaved product through a nickel column, and collecting eluent to obtain unlabeled FGF7 protein; 6) concentrating the unlabeled FGF7 protein, eluting with a molecular sieve Superdex 75 column, and collecting the eluate to obtain the high-activity FGF7 protein. The method can obtain the rmFGF7 protein with high bioactivity and high purity by 2-time nickel column purification, and provides a new way for the production of FGF7 protein.

Description

Preparation method of high-activity FGF7 protein

Technical Field

The invention relates to the field of molecular biology, in particular to a preparation method of a high-activity FGF7 protein.

Background

Fibroblast growth factor 7 (FGF 7), also known as keratinocyte growth factor KGF, was a cytokine first isolated from human embryonic lung fibroblasts by Rubin et al in 1989. Specifically binds to epithelial cell receptors, causes a series of signal transmission in cells, stimulates the proliferation, differentiation and migration of epithelial cells, and plays a unique role in the proliferation and damage repair of epithelial cells. Mouse and rat FGF7 homologous chromosome studies have shown that it is involved in the development of epithelial morphological functions, regeneration of wound epidermal cells, hair growth, and the like. FGF7 has many potential applications, such as chronic wound healing caused by metabolic diseases such as diabetes, application of bronchopulmonary dysplasia of premature infants, acute lung injury, treatment of acute liver injury and the like. FGF7 has been shown to have multiple mutants, and protein structure and function analysis indicated that deletion of the first 23 amino acids of the peptide chain did not reduce mitogenic activity of FGF 7. The America Amgen company expresses KGF mutant palifermin (Kepivence) with 23 amino acids deleted at the N terminal in Escherichia coli ^TM ) The traditional Chinese medicine composition is used for treating oral mucositis caused by radiotherapy and chemotherapy, and is approved by the FDA in the United states to be on the market in 12 months in 2004.

FGF7 has wide pharmacological activity, but the research and development progress of the medicament is severely limited due to the problems of low yield, instability, easy aggregation, high cost and the like of FGF7 protein in production.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provide a preparation method of FGF7 protein with high activity. The method of the invention utilizes the modified vector to express the FGF7 protein, and the SUMO label improves the expression quantity and solubility of the target protein, and is very convenient for purification. The protein FGF7 with high purity and good activity can be obtained by utilizing SUMO enzyme excision and subsequent simple purification, and a new way is provided for the production of FGF7 protein.

The technical scheme adopted by the invention is as follows:

a preparation method of high-activity FGF7 protein comprises the following steps:

(1) constructing pRSF-SUMO-rmFGF7 recombinant plasmid;

(2) transforming the pRSF-SUMO-rmFGF7 recombinant plasmid into host bacteria, carrying out induced expression, and collecting bacteria;

(3) carrying out ultrasonic crushing and centrifugation on the thalli, and collecting supernatant;

(4) eluting the supernatant through a nickel column, and collecting the eluent to obtain a primary purified substance;

(5) adding SUMO protease into the primary purified product, performing dialysis enzyme digestion, eluting the enzyme cut product through a nickel column, and collecting eluent to obtain unlabeled FGF7 protein;

(6) concentrating the unlabeled FGF7 protein, eluting with a molecular sieve Superdex 75 column, and collecting the eluate to obtain the high-activity FGF7 protein.

Preferably, the construction of the recombinant plasmid in step (1) comprises the following steps: inserting a 6 XHis-SUMO sequence between restriction enzyme sites Nco I and BamH I of a pRSFDuet-1 vector to obtain a pRSF-SUMO vector, wherein the 6 XHis-SUMO sequence is shown in SEQ ID NO. 1; the mouse FGF7 gene is inserted between enzyme cutting sites BamH I and Xho I of pRSF-SUMO vector to obtain pRSF-SUMO-rmFGF7 recombinant plasmid.

Preferably, the induction of expression in step (2) is by IPTG.

Preferably, the elution in step (4) is performed with a buffer containing 100 mM-200 mM imidazole, wherein the buffer has a composition of 25mM Tris, 300mM NaCl, and the solvent is water, and has a pH of 8.0.

Preferably, the elution in step (5) is performed using a buffer containing 0 mM-20 mM imidazole, wherein the buffer has a composition of 25mM Tris, 300mM NaCl, and the solvent is water, and has a pH of 8.0.

Preferably, the dialyzing enzyme in the step (5) is dialyzed overnight at 4 ℃ by adding precooled dialysis buffer solution with 100 times volume; the dialysis buffer solution consists of 25mM Tris, 300mM NaCl, water as solvent and pH8.0.

Preferably, the elution in step (6) is performed using PBS buffer consisting of 137mM NaCl, 2.7mM KCl, 4.3mM Na ₂ HPO ₄ ，1.4mM KH ₂ PO ₄ The solvent is water, and the pH value is 7.4.

The method of the invention utilizes the modified carrier and adds the His-SUMO label, thus improving the expression quantity and solubility of the target protein and being very convenient for purification. From experimental results, the purity of more than 95% can be achieved through the first nickel column, SUMO enzyme digestion and the second nickel column purification, molecular sieve purification shows that the obtained rmFGF7 protein is good in uniformity and free of dimer phenomenon, and 8.4mg of protein (6 mL in total of 1.4mg/mL protein solution) can be obtained from 1L of bacterial liquid. In conclusion, the method can obtain the rmFGF7 protein with high bioactivity and high purity by 2 times of nickel column purification.

Drawings

FIG. 1 shows the result of amplification of mouse FGF7 gene. The PCR product is shown as band 1 and Marker is shown as band 2.

FIG. 2 is a schematic diagram of the construction of recombinant plasmid pRSF-SUMO-rmFGF 7. The expression method comprises the steps of a, constructing an rmFGF7 expression plasmid pRSF-SUMO-rmFGF7, inserting a mature FGF7 sequence between modified pRSF-SUMO vector BamH I and Xho I restriction enzymes, and b, expressing rmFGF7 in a fusion protein form, wherein the N end of the fusion protein is provided with His, an SUMO tag and an SUMO restriction enzyme cutting site.

FIG. 3 is the first nickel column purification of rmFGF7 containing a His-SUMO tag.

FIG. 4 shows SUMO enzyme digestion and secondary nickel column purification.

FIG. 5 shows molecular sieve purification.

FIG. 6 shows silver staining and Western blotting detection.

FIG. 7 shows the activity assay of purified rmFGF 7.

Detailed Description

The following examples are further illustrative of the present invention and are not intended to be limiting thereof.

Example 1

1. Material

Coli strains DH5 α, BL21(DE3) were purchased from Tiangen Biochemical technology (Beijing) Ltd. The pRSFDuet-1 plasmid was stored in the laboratory. Mouse FGF7 antibody was purchased from santa cruz. Restriction endonucleases Nco I, BamH I, Xho I, pfu DNA polymerase, T4 DNA ligase, protein molecular weight Marker were purchased from Thermo Scientific. The DNA rapid purification and recovery kit, the gel recovery kit and the kanamycin sulfate are purchased from Shanghai biological engineering technology, Inc. Trizol and reverse transcription kits were purchased from Bao bioengineering (Dalian) Inc. isopropyl-beta-D-thiogalactoside (IPTG) was purchased from guangzhou seiko biotechnology limited. Other biochemical reagents are domestic analytical purifiers. The primers used in this work were synthesized by Shanghai Biotechnology, Inc. (Table 1).

TABLE 1 primer sequences

Primer name	Primer sequence (5'-3')
		pRSF-sumo F	CATGCCATGGGCAGCAGCCATCATCA
pRSF-sumo R	CGGGATCCACCAATCTGTTCTCTGTGA
		pRSF-sumo-FGF7 F	CGGGATCCTGCAATGACATGAGT
pRSF-sumo-FGF7 R	CCGCTCGAGTTAGGTTATTGCCATAGGA

2. Vector construction

The synthesized 6 XHis-SUMO sequence (shown in SEQ ID NO. 1) is used as a template, primers pRSF-SUMO F/pRSF-SUMO R are used for PCR amplification and enzyme digestion by Nco I and BamH I, T4 DNA ligase is used for ligation and transformation of Escherichia coli DH5 alpha, a single colony is picked up and cultured overnight at 37 ℃, a plasmid is extracted and analyzed through sequencing, the SUMO sequence is successfully inserted between the enzyme digestion sites Nco I and BamH I of the pRSFDuet-1 vector, and the modified vector is named as pRSF-SUMO. Mouse lung RNA was extracted with TRIzol, cDNA was synthesized by reverse transcription, and mouse FGF7 gene was PCR-amplified using mouse FGF 7-specific upstream and downstream primers (upstream primer: 5'-CGGGATCCTGCAATGACATGAGT-3', downstream primer: 5'-CCGCTCGAGTTAGGTT ATTGCCATAGGA-3') using cDNA as a template. FGF7 gene is inserted between BamH I and Xho I endonucleases of a modified vector pRSF-SUMO to construct an FGF7 prokaryotic expression vector, after colony culture of transformed escherichia coli DH5 alpha, a primer pRSF-SUMO-FGF 7F/pRSF-SUMO-FGF 7R is used for PCR identification to be positive, and further DNA sequencing is used for verification to be positive clone, so that prokaryotic expression plasmid of mouse FGF7 is obtained and named as pRSF-SUMO-rmFGF 7.

The result shows that a product is obtained by PCR amplification by using mouse FGF7 specific primers and mouse cDNA as a template, and a specific band is found to exist around 500bp by electrophoresis, which is consistent with the experimental design (figure 1). The plasmid is inserted into a modified pRSF-SUMO vector after double enzyme digestion by BamH I and Xho I endonucleases, is positive through PCR identification after colony culture of the transformed DH5 alpha, is further confirmed to be positive clone through sequencing, and obtains prokaryotic expression plasmid of mouse FGF7, and the construction schematic diagram of the plasmid is shown in figure 2.

3. Protein expression and nickel column affinity purification

The expression vector pRSF-SUMO-rmFGF7 was transformed into E.coli BL21(DE3), a single clone was picked up and cultured overnight at 37 ℃ in 5mL of LB medium containing kanamycin resistance, inoculated into fresh LB medium at a ratio of 1:100, cultured until OD600 reached 0.8-1.0, and induced to express at 37 ℃ for 4 hours by adding IPTG at a final concentration of 1 mM. 1mL of the bacterial culture was collected, the cells were collected by centrifugation at 5000rpm, the pellet was resuspended in 100. mu.L of 1 Xloading buffer (25mM Tris, 300mM NaCl, solvent water, pH8.0), boiled at 100 ℃ for 8min, 10. mu.L of the pellet was centrifuged and subjected to SDS-PAGE to analyze the expression of rmFGF 7.

Glycerol bacteria expressing rmFGF7 were inoculated into 100mL of LB medium, shake-cultured overnight at 37 ℃ and transferred into 2 flasks containing 1000mL of LB medium at a ratio of 1: 100. Shaking culturing at 37 deg.C until OD600 reaches 0.8-1.0, adding 0.3mM IPTG inducer, inducing at 37 deg.C for 4 hr, and centrifuging to collect thallus. 1L of the collected culture medium was resuspended in 50mL Buffer A (25mM Tris, 300mM NaCl, solvent water, pH8.0) and disrupted by sonication after adding protease inhibitor (1mM PMSF), and the supernatant was collected by centrifugation at 12000rpm at 4 ℃ for 30 min. The supernatant was added to a nickel column equilibrated with Buffer a and the flow-through was discharged. The fractions were eluted with Buffer A and Buffer B (25mM Tris, 300mM NaCl, 500mM imidazole in water, pH8.0) containing 20mM, 50mM, 100mM, 200mM imidazole, and the eluates of the fractions were collected. Taking the whole bacterial liquid cultured by expressing the rmFGF7 strain, supernatant obtained by inducing, carrying out ultrasonic crushing and centrifuging on the bacterial liquid, flow-through liquid and 20 mu L of each eluent, adding 5 mu L of 5 multiplied loading buffer solution (25mM Tris, 300mM NaCl, water serving as a solvent and pH8.0) for resuspension, boiling the samples at 100 ℃ for 8min, centrifuging, taking 10 mu L of the mixture, carrying out SDS-PAGE electrophoresis and Coomassie brilliant blue staining, and obtaining the primarily purified rmFGF7 protein with His-SUMO label.

The results show that the supernatant after centrifugation was subjected to a first nickel column purification, enabling elution at 100-.

4. SUMO tag excision and secondary nickel column purification

The primarily purified rmFGF7 protein with His-SUMO tag was collected, SUMO protease (also known as ULP1 enzyme) (prepared in the laboratory) was added, and the mixture was packed into dialysis bags, which were placed in a pre-cooled 100-fold volume of dialysis Buffer (Buffer A) and dialyzed overnight at 4 ℃. The digested sample was added to a Buffer A equilibrated nickel column and the flow through was discharged. Then eluting through Buffer A, Buffer A containing 20mM imidazole and Buffer B, and collecting each part of eluent. Taking 20 mu L of sample solution before enzyme digestion, sample solution after enzyme digestion, flow-through solution and each eluent respectively, adding 5 mu L of 5 Xloading buffer solution (25mM Tris, 300mM NaCl, solvent water, pH8.0) for heavy suspension, boiling the samples for 8min at 100 ℃, centrifuging, taking 10 mu L of the samples for SDS-PAGE electrophoresis and Coomassie brilliant blue staining, and obtaining the high-purity unlabeled rmFGF7 protein.

The results show that the collected primarily purified samples are cut by dialysis enzyme, and the cut samples are 3 main bands, the top is SUMO enzyme, the middle is rmFGF7 of the cutting label, and the bottom is SUMO-tag. The enzyme-digested sample is purified by a second nickel column, and the purity of the sample purified by the second nickel column is very high as shown in the SDS-PAGE result (FIG. 4).

5. Molecular sieve fine purification

Further purification was performed using a molecular sieve Superdex 75 column. The second nickel column purified rmFGF7 protein sample was concentrated to 2mL using an ultrafiltration tube and injected into PBS (137mM NaCl, 2.7mM KCl, 4.3mM Na) ₂ HPO ₄ ，1.4mM KH ₂ PO ₄ Solvent is water, pH7.4), collecting the eluate of the peak, and performing SDS-PAGE electrophoresis detection. The FGF7 protein obtained was purified and analyzed for purity by silver staining, and the target protein was further confirmed by WB.

The results show that the protein sample is monomer and has good homogeneity when purified by molecular sieve, and the SDS-PAGE result shows that the purified rmFGF7 protein has high purity (figure 5). The purified rmFGF7 protein is silver stained after SDS-PAGE, and the result shows that the purity of the recombinant protein reaches more than 95%, and the recombinant protein can generate positive reaction with the mouse FGF7 antibody, as shown in figure 6.

6. Activity assay of purified rmFGF7

MCF-7 and HaCaT cell lines were cultured in DMEM containing 10% Fetal Bovine Serum (FBS) at 37 ℃ in 5% CO ₂ Culturing in an incubator. Cells were trypsinized and counted up to log phase, added to 96-well plates at 5000 cells per well, cultured overnight, and then replaced with fresh DMEM medium containing 1% FBS for starvation culture for 24 h. The original medium was aspirated, and the purified recombinant protein rmFGF7 was serially diluted with 1% FBS-containing DMEM medium (rm)FGF7 was added at a concentration of 0.1, 1, 10, 20, 50, 100, 200, 1000ng/mL) to the cell culture plates (100. mu.L/well) at 5 replicates per concentration, and 1% FBS in DMEM medium was added to the control group. After 48h of culture, adding CCK8 or MTT to incubate for 30min-2h, and reading the light absorption value of 450nm or 490nm by using a microplate reader.

The results of the activity assay of purified rmFGF7 using MTT and CCK8 are shown in FIG. 7. The result shows that the rmFGF7 expressed by the escherichia coli can stimulate the proliferation of epithelial cells and keratinocytes and has remarkable mitogenic activity.

The characteristic that FGF7 is only combined with FGFR2IIIb specifically expressed by epithelial cells enables the administration of the drug to be safer, so the drug has wide prospect in clinical application. Researchers have expressed hFGF7 using yeast, plant and mammalian cells so far, but there are problems of low expression level, inclusion body, complicated purification, and cost. The method of the invention utilizes the modified carrier and adds the His-SUMO label, thus improving the expression quantity and solubility of the target protein and being very convenient for purification. From experimental results, the purity of more than 95% can be achieved through the first nickel column, SUMO enzyme digestion and the second nickel column purification, molecular sieve purification shows that the obtained rmFGF7 protein is good in uniformity and free of dimer phenomenon, and 8.4mg of protein (6 mL in total of 1.4mg/mL protein solution) can be obtained from 1L of bacterial liquid. In conclusion, the method can obtain the rmFGF7 protein with high bioactivity and high purity by 2 times of nickel column purification.

The above is only a preferred embodiment of the present invention, and it should be noted that the above preferred embodiment should not be considered as limiting the present invention, and the protection scope of the present invention should be subject to the scope defined by the claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and should be considered to be within the scope of the invention.

Claims (7)

1. A preparation method of high-activity FGF7 protein is characterized by comprising the following steps:

(1) constructing pRSF-SUMO-rmFGF7 recombinant plasmid;

(2) the pRSF-SUMO-rmFGF7 recombinant plasmid is transformed into host bacteria, induced expression is carried out, and bacteria are collected;

(3) carrying out ultrasonic crushing and centrifugation on the thalli, and collecting supernatant;

(4) eluting the supernatant through a nickel column, and collecting the eluent to obtain a primary purified substance;

(5) adding SUMO protease into the primary purified product, performing dialysis enzyme digestion, eluting the enzyme cut product through a nickel column, and collecting eluent to obtain unlabeled FGF7 protein;

(6) concentrating the unlabeled FGF7 protein, eluting with a molecular sieve Superdex 75 column, and collecting the eluate to obtain the high-activity FGF7 protein.

2. The method for preparing FGF7 protein according to claim 1, wherein the recombinant plasmid obtained in step (1) is prepared by the steps of: inserting a 6 XHis-SUMO sequence between restriction enzyme sites Nco I and BamH I of a pRSFDuet-1 vector to obtain a pRSF-SUMO vector, wherein the 6 XHis-SUMO sequence is shown in SEQ ID NO. 1; the mouse FGF7 gene is inserted between enzyme cutting sites BamH I and Xho I of pRSF-SUMO vector to obtain pRSF-SUMO-rmFGF7 recombinant plasmid.

3. The method for preparing FGF7 protein according to claim 1, wherein the inducing expression in step (2) is performed by IPTG.

4. The method of claim 1, wherein the elution in step (4) is performed with a buffer containing 100 mM-200 mM imidazole, wherein the buffer comprises 25mM Tris, 300mM NaCl, and water, and the pH is 8.0.

5. The method for preparing FGF7 protein with high activity according to claim 1, wherein the elution performed in step (5) is performed using a buffer containing 0 mM-20 mM imidazole, wherein the buffer comprises 25mM Tris, 300mM NaCl, water as a solvent, and pH 8.0.

6. The method of claim 1, wherein the step (5) of dialyzing the high activity FGF7 protein comprises adding pre-cooled 100 volumes of dialysis buffer at 4 ℃ overnight; the dialysis buffer solution consists of 25mM Tris, 300mM NaCl, water as solvent and pH8.0.

7. The method for preparing a FGF7 protein of claim 1, wherein said elution of step (6) is performed by using PBS buffer consisting of 137mM NaCl, 2.7mM KCl, and 4.3mM Na ₂ HPO ₄ ，1.4mM KH ₂ PO ₄ The solvent is water, and the pH value is 7.4.

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