ABSTRACT
Enteropeptidase is a duodenum serine protease that triggers the activation of pancreatic enzymes by remarkably specific cleavages after lysine residues of peptidyl substrate (Asp)4-Lys. This high specific cleavage makes the enzyme a widely used biotechnological tool in laboratory researches and industrial scale. Previous studies both in small and large scales were showed low expression and miss-folding of the expressed protein. In this study, the DNA sequence encoding the light chain (catalytic subunit) of bovine enteropeptidase (EPL) was subcloned into plasmid pET-32b, downstream to the DNA encoding the fusion partner thioredoxin immediately after the EPL cleavage site. SHuffle® T7 Express was selected as an expression host due to the ability to promote proper folding and correction of the mis-oxidized bonds. Expression and purification of protein was performed, and the result of biological activity confirmed that the active EPL was obtained. Optimization of protein expression conditions was accomplished by response surface methodology for significant factors including induction temperature, duration of induction, inducer concentration and OD600 of induction. The best conditions were achieved in 1.05 mM IPTG at OD600 of 0.6 for seven h incubation at 26.5 °C, and a high level of protein expression was obtained in the optimized condition.
