Construction of hFAK Gene-recombinant Plasmids and its Protein Expression

Objective To construct the recombinant plasmids of human hFAK gene and identify its protein expression.Methods Total RNA was extracted from human gastric cancer SGC-7901 cells.The hFAK coding sequence was amplified by polymerase chain reaction(PCR)method and cloned into pCDNA3.1/Flag and pGEX-4T-2 vectors respectively.IPTG-induced GST-hFAK protein expression in BL21 cells and protein purification were identified by SDS-PAGE.The expression and localization of the pCDNA3.1/Flag-hFAK recombinant plasmid in SGC-7901 cells was proved by western blot and immunofluorescence.Results hFAK had been cloned into pGEX-4T-2 and pCDNA3.1/Flag successfully.The length of the fragment was about 3200 bp.The expression of IPTG induced GST-hFAK and protein purification were identified by SDS-PAGE and commassie blue staining.The Flag-hFAK expression in SGC-7901 cells was identified by western blot with a molecular weight of 130 kD.Conclusion The full length sequence of hFAK was successfully cloned into prokaryotic and eukaryotic expressing vectors,and the expression of these plasmids were identified.