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.

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The Dunaliella salina enolase gene (DsENO) had been cloned using Rapid Amplification of cDNA Ends methods. Recombinant D. salina enolase was over-expressed in E. coli BL21 and purified. Native polyacrylamide gel electrophoresis of recombinant enolase indicated that it forms a homo-dimer in the native state. Polyclonal antiserum against purified recombinant D. salina enolase was raised in a rabbit. The enolase activity of DsENO was examined in Kluyveromyces lactis enolase null mutant and DsENO partly complemented the enolase null mutant of K. lactis Rag À phenotype. The protein level of D. salina enolase was determined under various conditions. The enolase protein level decreased by more than 50% after between 1.5-and 3-h exposure to hyperosmotic salt stress. This was confirmed by the enolase activity assay. It is suggested that enolase takes part in glycerol synthesis, which can balance the external salt concentration. Under heat-shock treatment, induction of enolase was observed, which suggested that D. salina enolase may contribute to its thermal tolerance.

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