Cloning and prokaryotic expression of full-length gene of human granzyme A

Objective: To clone human granzyme A (GzmA) gene, express in E. coli and preliminarily optimize the condition for expression. Methods: Human GzmA gene was amplified by RT-PCR and inserted into prokaryotic expression vector pET24a (+). The constructed recombinant plasmid pET24a-GzmA was transformed to E. coli BL21 (DE3) and induced with IPTG. The expressed product was identified by SDS-PAGE and Western blot. The temperature, IPTG concentration and time for induction as well as the A ₆₀₀ value of bacterial liquid when the induction was started were optimized. Results: Restriction analysis and sequencing proved that GzmA gene with correct sequence was inserted into vector pET24a(+). The expressed recombinant protein, with a relative molecular mass of about 29 000, showed specific binding to mouse anti-His monoclonal antibody. The optimal temperature for induction of recombinant E. coli was 37°C. However, IPTG concentration, time for induction and the A ₆₀₀ value of bacterial liquid when the induction was started showed little effect on expression level of recombinant protein. Conclusion: Human GzmA gene was successfully cloned and expressed in E. coli.