Molecular chaperones and solubility tags: A 44-fold increase in recombinant fibEase yield for therapeutic...

Following codon optimization, the fibE of marine origin achieved successful intracellular expression in Escherichia coli. To enhance production, a combinatorial strategy involving codon optimization, solubility-enhancing tags, molecular chaperones, and process optimization was implemented. After optimization, the fibrinolytic enzyme activity of recombinant strain reached 12294 U/mL per unit volume of fermentation broth, which was 44 times that before optimization. Differential expression analyses between recombinant E. coli with or without molecular chaperones enabled functional validation. Molecular chaperones were shown to promote correct folding and soluble expression of the target fibEase. Following this, the molecular chaperone and the solubilizing tag simultaneously dissociated, minimizing possible losses caused by thrombin-mediated cleavage and tag removal during the second purification step. As a serine protease reliant on metal ions, fibEase did not induce hemolysis in vitro and specifically degraded fibrin directly. The combinatorial optimization described in this study might be utilized for expression of other hard-to-express proteins. Finally, the superior enzymatic properities suggest potential preventive or therapeutic applications in thrombosis.