Aims: The objective of this study is to actively express a novel fibrinolytic enzyme, subtilisin DFE (douchi fibrinolytic enzyme), in Escherichia coli.
Methods and results: The DNA fragments encoding pro-subtilisin DFE was amplified and cloned into the vector pET32a to obtain N-terminal Trx fusion expression plasmid. The recombinant subtilisin DFE was successfully expressed and processed in the soluble fraction of E. coli BL21(DE3) in a similar fashion as the endogenous one of Bacillus amyloliquefaciens DC-4, resulting in an active enzyme. Moreover, active enzyme can also be refolded from inclusion body.
Conclusions: Active subtilisin DFE can be expressed and processed in E. coli.
Significance and impact of the study: This study provides evidences that subtilisin DFE can be actively expressed in E. coli and the pro-peptide is essential for guiding the proper folding into the active conformation. As such, large quantities of recombinant subtilisin DFE can be produced for pharmacological and clinical research.