Construction and Optimization of Prethrombin-2 Human Genes in E. coli for the Production of Active Thrombin

摘要

Prothrombin-2 is a thrombin precursor, which plays an essential role in the conversion of fibrinogen to fibrin during the blood clotting process. In this study, we designed and constructed the human prethrombin-2 (hPT2) gene. hPT2 gene fused with a tag at the n terminal position, which contains the sequence intein followed by chitin binding domain (CBD) that useful for the purification process. The digestion process of hPT2 tag induced by changing its pH/ temperature. The hPT2 codon that used in this study designed according to the preference codon of E. coli. hPT2 gene was created using the OPTIMIZER software with the addition of two side restriction at the 5' and 3', BamHI and XhoI respectively then were cloned by using the pMAT vector in E. coli. Moreover, hPT2 fragment ligated into an expression vector, pTWIN1, for E. coli. The result from the characterization hPT2 gene in pTWIN1 that using DNA sequencing method shown that the designed hPT2 gene cloned. Thus, the cloned hPT2 gene used as a precursor for PT2 expression in the E. coli host.