A Convenient and Effective Approach for Gene Synthesis of Human Platelet Factor-4
- VernacularTitle:一种合成人血小板因子4基因的简便有效方法
- Author:
Xiaoping WU
;
Zhijian SU
;
Qing ZHENG
;
Sixian WU
;
Hua XU
;
Wen ZHAO
;
Xiaokun LI
- Publication Type:Journal Article
- Keywords:
Human platelet factor-4;
Gene synthesis;
Gene expression
- From:
Journal of China Pharmaceutical University
2005;(6):590-593
- CountryChina
- Language:Chinese
-
Abstract:
AIM:To synthesize the human platelet factor-4(hPF4) gene with a convenient and effective approach, and high express the hPF4 gene in E. coli BL21 (DE3). METHODS: According to the primary structure of hPF4, the nucleotide sequence was synthesized using touch-down PCR method. The resultant gene fragment containing EcoR Ⅰ and Xho Ⅰ overhangs at 5' and 3' ends was cloned into the expression vector pGEX-4T-1 to construct the recombinant plasmid pGEX-4T-1-hPF4,which was then transformed into the E. coli strain BL21 (DE3). RESULTS: hPF4 gene was successfully synthesized by touchdown PCR method. A fusion protein composed of glutathione S-transferase (GST) and the recombinant hPF4 was expressed in BL21(DE3) by IPTG induction. The expression level of the fusion protein in E. coli was about 30% of the total cellular protein. CONCLUSION: Touch-down PCR may provide a convenient and effective approach to obtain other target genes. The expressed fusion protein forms the inclusion bodies, providing sufficient material for further purification and biological activities process.
