Cloning and high-efficiency expression of nattokinase gene

In this study,the nattokinase gene with signal peptide,propeptide and mature peptide was cloned and expressed in Escherichia coli,and the induction and expression parameters were optimized.The gene was amplified with genomic DNA extracted from Bacillus subtillis as template by RT-PCR,digested and ligated with expression vector-pET28a to construct recombinant plasmid pET28a-NK which was transformed into E coli strain BL21(DE3).Induction conditions such as time,temperature and IPTG concentration were studied,and the high-est fibrinolytic activity was obtained and it could reach 81.73 U?mL-1 when recombinant strain BL21(DE3)-NK was cultivated at 20℃ for 20 h with IPTG at 0.4 mmol?L-1.An expected protein band about 38 kDa was observed by SDS-PAGE.