Construction and induction of GST-hCAP fusion protein expression in prokaryotic cells

Objective: To construct GST-tagged human c-Cbl-associated protein(GST-hCAP) fusion protein expression vector and induce its expression in Escherichia coli(E.coli).Methods: Total mRNA was extracted from COS-1 cells and cDNA was synthesized by reverse transcription.The hCAP coding sequence was amplified by polymerase chain reaction(PCR) and subcloned into pGEX-5X-1 vector.The insert was identified by restriction enzyme digestion and DNA sequencing.pGEX-5X-1-hCAP and pGEX-5X-1 were transformed into E.coli BL21,induced by IPTG and identified by SDS-PAGE and Western blot.Results: The prokaryotic expression plasmid pGEX-5X-1-hCAP was successfully constructed and confirmed by enzyme digestion and sequencing.The GST-hCAP fusion proteins were identified by Western blot.Conclusion: The prokaryotic expression plasmid of hCAP is successfully constructed and the expression of fusion proteins in E.coli is confirmed.This study provides the basis for the further research on purifying CAP protein and the biological function of CAP.