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Human FGF21 mutant gene and method for preparing recombinant human FGF21 protein

A technology of FGF21 and mutant gene, which is applied in the application field of human fibroblast growth factor, mutant gene, and the preparation of recombinant human FGF21 protein, can solve the problems of low stability, high cost, poor specificity, etc., and achieve high stability , low cost, good specificity

Inactive Publication Date: 2011-02-09
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The existing proteases or protein factors used to cut tags from fusion proteins mainly have defects such as poor specificity, low stability, and inability to maintain high activity in a

Method used

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  • Human FGF21 mutant gene and method for preparing recombinant human FGF21 protein
  • Human FGF21 mutant gene and method for preparing recombinant human FGF21 protein
  • Human FGF21 mutant gene and method for preparing recombinant human FGF21 protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Construction and expression of embodiment 1mutFGF21 gene

[0043] 1. Construction of recombinant expression plasmids

[0044] The SUMO sequence with 6×His tag sequence (Nde I and BsaI restriction sites introduced at both ends, respectively) was cloned into the pMD18-T vector to construct the pMD18-T-SUMOHis plasmid. The human FGF21 sequence with hydroxylamine sites (respectively introduced BsaI and BamHI restriction sites at both ends) was cloned into pMD18-T vector to construct pMD18-T-Asn-Gly-hFGF21 plasmid. The SUMOHis fragment obtained by digesting pMD18-T-SUMOHis with Nde I and BsaI was ligated with the Asn-Gly-hFGF21 fragment obtained by digesting pMD18-T-Asn-Gly-hFGF21 with BsaI and BamHI, and the ligated product was cloned again To the pET30a(+) digested with Nde I and BamHI, the recombinant expression plasmid pET30a(+)-SUMOHis-Asn-Gly-hFGF21 was obtained. For the construction process, see figure 1 .

[0045] 2. Site-directed mutation of hFGF21 gene

[0046]...

Embodiment 2

[0080] Preparation and purification of embodiment 2 human recombinant FGF21 fusion protein

[0081] 1. Conversion

[0082] Melt Rosetta-competent on ice, add 1 μl (10ng) recombinant expression plasmid, place on ice for 30 minutes, heat shock at 42°C for 45 seconds, add 200μl LB liquid (without Kan), shake at 37°C for 45 minutes, spread on the plate, and invert at 37°C Incubate overnight.

[0083] 2. Express

[0084] (1) Activation: pick a single colony in 10ml LB (50μg / ml Kan), shake overnight at 37°C;

[0085] (2) Secondary activation: 1 / 100 inoculation (1000ml inoculated with 10ml, 50μg / mlKan), shake at 37°C for 2-3 hours to OD 600 =0.3;

[0086] (3) Induction:

[0087] Two groups were set up and induced according to the following induction conditions:

[0088] Test group: Temperature: 37°C,

[0089] IPTG final concentration: 0.25mmol / L,

[0090] Speed: 100r / min,

[0091] Shaking time: 3 hours;

[0092] Control group: Temperature: 37°C,

[0093] Speed: 100r / min, ...

Embodiment 3

[0104] Example 3 Hydroxylamine Cleavage of Human Recombinant FGF21 Fusion Protein of the Present Invention and Purification of Human Recombinant FGF21

[0105] 1. Test material

[0106] Test sample: the human recombinant FGF21 fusion protein prepared and purified in Example 2;

[0107] Reagents: Hydroxylamine cleavage buffer.

[0108] 2. Test methods and results

[0109] (1) Exploration of fusion protein hydroxylamine cleavage concentration

[0110] Concentrate the fusion protein (concentration: 1 mg / ml) 5 times to a concentration of 5 mg / ml, add 2× hydroxylamine cleavage buffer at a volume ratio of 1:1, 1:2, 1:3, 1:4, 1:5 After reacting at 30°C for 1 hour, each system was added to PBS pH 7.2 to the volume before protein concentration, and samples were taken for detection by 15% SDS-PAGE to analyze the optimal concentration of hydroxylamine cleavage buffer. see results Figure 11 .

[0111] (2) Purification of human recombinant FGF21

[0112] After the fusion protein wi...

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Abstract

The invention discloses a human FGF21 mutant gene and a method for preparing a recombinant human FGF21 protein. The mutation is carried out on a human FGF21 gene, the base sequence after the mutation is as shown in SEQ IDNO.1, and the expression level of the gene after the mutation in prokaryocytes is significantly improved. The invention further provides a method for improving the expression level of the human FGF21 protein, which comprises the following steps: sequentially connecting a 6 multiplied by His tag gene, an SUMO tag gene, a hydroxylamine cleavage site gene and the human FGF21 mutant gene together, and then obtaining a gene as shown in SEQ ID NO.3; connecting the obtained gene with an expression vector, and obtaining a recombinant expression vector; transforming into host cells; culturing, inducing the expression of the recombinant protein, collecting and purifying the expressed protein, carrying out hydroxylamine cleavage, and further purifying. The method has the advantages of good specificity, high stability, capability of keeping the cleavage activity in a reaction environment system in a wide range, low preparation cost and the like.

Description

technical field [0001] The present invention relates to a mutated gene, in particular to a human FGF21 mutated gene and human fibroblast growth factor (hFGF21) encoded by the gene. The present invention also relates to the use of the human FGF21 mutated gene in preparing recombinant human FGF21 protein The application belongs to the field of preparation of human FGF21 recombinant protein. Background technique [0002] In 2000, Tetsuya Nishimura et al first reported the human FGF21 gene. The gene is located in the 5'flanking region of the human α1,2-fucosyltransferase gene, and encodes a 209-amino acid polypeptide that is specifically expressed in the liver. The first 28 amino acids at the N-terminal of the protein are signal peptides. Therefore, FGF21 can be secreted extracellularly. Through sequence comparison analysis, human FGF21 belongs to the FGF19 subfamily of the FGF family, which includes FGF15, FGF19, FGF21, and FGF23. FGF21 has the highest homology with FGF19 in ...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/12C07K14/50C12N1/19C12N1/21C12N5/10C12N1/15
Inventor 任桂萍李德山高华山刘铭瑶王文飞
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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