Expression and purification methods of fusion protein containing recombinant human fibroblast growth factor 21

A human fibroblast and fusion protein technology, applied in the field of biomedicine, can solve the problems of low purity and protein loss, and achieve the effects of improving protein purity, reducing protein loss, and increasing protein expression.

Inactive Publication Date: 2016-04-20
INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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Problems solved by technology

[0004] According to reports, the current systems for expressing the protein include SUMO system and yeast system. The expression of the protein in the SUMO system is purified by His-tag affinity chromatography. The expression of the protein in the yeast system is purified by ion-exchange column chromatography. , after purification with the above method, not only the protein loss is large but also the purity is not high

Method used

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  • Expression and purification methods of fusion protein containing recombinant human fibroblast growth factor 21
  • Expression and purification methods of fusion protein containing recombinant human fibroblast growth factor 21
  • Expression and purification methods of fusion protein containing recombinant human fibroblast growth factor 21

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Construction of hFGF21 Gene Recombination Plasmid of pGEX-5X-1

[0036] 1.1PCR to obtain the target fragment of hFGF21 mature peptide

[0037] Human liver cell cDNA was used as template plus downstream primers for PCR amplification. PCR system: template, upstream and downstream primers 1 μl each, PCRmastermix 12.5 μl, H2O 9.5 μl; PCR conditions: 94°C 10min, 94°C 30s, 52°C 30s, 72°C 1min35 cycles, 72°C 7min, 4°C 12min, PCR product It was identified and recovered by 1.4% agarose gel.

[0038] 1.2 Construction of vector PGEX-5X-1-hFGF21

[0039] 1.2.1 Ligation of plasmid PGEX-5X-1 and target fragment hFGF21

[0040] (1) Carry out corresponding enzyme digestion on the plasmid and PCR product first, restriction enzyme digestion system: 5 μl of PGEX-5X-1 plasmid, 2 μl of 10xQuikCutGreenBuffer, 1 μl of restriction endonuclease EcoRI and BamHI, H2O11 μl; 12 μl of hFGF21 PCR product fragment, 2 μl of 10xQuikCutGreenBuffer, restriction Endonucleases EcoRI and BamHI 1 μl each,...

Embodiment 2

[0064] Induced Expression of hFGF21 Mature Peptide

[0065] 2.1. Transform the PGEX-5X-1-hFGF21 recombinant plasmid into BL21 competent cells.

[0066] 2.2 Optimizing induction conditions

[0067] (1) Take a test tube containing 5ml of sterilized LB liquid medium, add 5μl of 100mg / ml Amp in aseptic operation and shake well, pick out monoclonal bacteria in a good growth state and put them into the test tube, shake at a constant temperature of 37°C and 250rpm / min overnight;

[0068] (2) Take a test tube containing 5 ml of sterilized LB liquid medium, add 5 μl of 100 mg / ml Amp in aseptic operation and shake well, take 50 μl of the bacterial solution and transfer it to the test tube;

[0069] (3) Shake the test tube in a constant temperature shaking box at 37°C and 250 rpm / min for about 1.5-2 hours. When the OD value is measured to be 0.3, add 1mol / LIPTG5μl to start the shaking induction;

[0070] (3) At 0, 2, 4, and 6 hours after adding the inducer, sample 1ml of the bacterial ...

Embodiment 3

[0079] Purification of recombinant proteins

[0080] 3.1 Treatment and renaturation of inclusion bodies

[0081] 3.1.1. Inclusion body treatment

[0082] (1) Add pre-cooled 30ml of PB buffer solution with a pH value of 8.0 to each 50ml tube of bacterial pellet to resuspend the bacteria, and ultrasonically disrupt for 30 minutes with an ultrasonic breaker. During ultrasonic disruption, the bacterial solution should be kept at a low temperature of 2-8°C;

[0083] (2) After crushing, centrifuge at 7500rpm for 10min at 4°C, sample 100μl of the supernatant, dissolve the sediment in 100μl of water, add 5μl of 5xSDS-PAGE loading buffer to boil for 10min, and run protein gel to test whether the protein is expressed in the inclusion body.

[0084] (3) After sonication, the bacteria were precipitated and pre-cooled with about 10ml of Triton to resuspend the bacteria, sonicated for 1 minute, placed at 4°C for 30 minutes, centrifuged at 7000rpm at 4°C for 15 minutes, and washed with impu...

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Abstract

The invention provides expression and purification methods of a fusion protein containing a recombinant human fibroblast growth factor 21 (hFGF21). In an expression process, the fusion protein is the fusion protein of GST protein and recombinant human fibroblast growth factor 21, is transferred into BL21 after being constructed into a plasmid with a GST tag, is subjected to IPTG induced expression, is greatly expressed in an inclusion body manner and is purified through ion-exchange column chromatography and a GST affinity column, and therefore a large quantity of high-purity proteins are obtained. The expression and purification methods have the beneficial effects that by expressing hFGF21 proteins by virtue of a GST system and optimizing induction conditions, the expression quantity of the proteins is substantially increased; by virtue of purification through the ion-exchange column chromatography and secondary purification through the GST affinity column, the loss of the proteins can be reduced as much as possible, and the purity of the proteins can be effectively improved; and the methods have important practical significance to the development of druggability of the hFGF21 proteins.

Description

technical field [0001] The invention belongs to the field of biomedicine, in particular to a method for expressing and purifying recombinant human fibroblast growth factor 21 fusion protein. Background technique [0002] According to the statistics of WHO in 2011, there are 366 million diabetic patients in the world, and 4.6 million people die of this disease every year; in 2012, the report of "China Chronic Disease Surveillance and Diabetes Special Survey" shows that there are 97 million diabetic patients in China , of which T2DM patients account for about 95%, and it is increasing year by year. At present, there is no definite and effective treatment method to cure the disease. The clinically used drugs such as insulin and thiazolidinediones have not achieved the purpose of treating both symptoms and root causes. According to the administration time point, they only have a short-term hypoglycemic effect and cannot meet the body's needs. Circadian rhythm regulation mechani...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/70A61K38/18A61K47/48A61P3/10A61P3/04
CPCC07K14/50A61K38/00C07K2319/23C12N15/62
Inventor 鲁帅尧张莹李鸿钧马开利陈瑜和占龙吴正存马进胡婧雯柳光龙
Owner INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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