Prokaryotic Expression and Functional Analysis of Recombinant Human bFGF

Dou Yuanyuan^1,2, Lin Yan^2,3, Gao Xiangzheng², Wang Yanfang², Wang Xiaoyin^1,2, Wang Tianyun^1,2*

(¹Department of Biochemistry and Molecular Biology, Xinxiang Medical University, Xinxiang 453000, China; ²International Joint Research Laboratory for Recombinant Pharmaceutical Protein Expression System of Henan, Xinxiang 453000, China; ³Department of community nursing, School of Nursing, Xinxiang Medical University, Xinxiang 453000, China)

Abstract:

In the present study, we studied the prokaryotic expression and purification of recombinant human basic fibroblast growth factor (bFGF), and analyzed its functions. Firstly, according to the bFGF gene sequence, the codon was optimized and the prokaryotic expression vector of pET-28a was constructed. And protein expression was induced by IPTG. Secondly, The expression of bFGF was determined by SDS-PAGE electrophoresis and confirmed by SDS-PAGE. Finally, NIH3T3 cells, HEK293 cells and CHO cells were cultured and CCK-8 assay was performed to detect protein activity. The results showed that the prokaryotic expression vector was successfully constructed. The target protein bFGF was successfully expressed under the induction of 1 mmol/L IPTG. The expression amount was about 32% of the bacterial protein and the protein purity was about 96%. The results of the activity test showed that the ED50 was 5.97 ng/mL, 4.21 ng/mL, and 6.71 ng/mL, which could effectively promote the proliferation of NIH3T3 cells, HEK293 cells, and CHO cells. This study demonstrates that the human bFGF protein was successfully expressed by prokaryotic expression system and its activity was high.

CSTR: 32200.14.cjcb.2019.07.0016