Comparison of Periplasmic and Cytoplasmic Expression of Bovine Enterokinase Light Chain in E. coli.

Enterokinase enzyme is widely used in production of recombinant proteins. This enzyme is isolated from the intestine and recognizes a specific cleavage site (X↓LYS-ASP4). Several studies have been performed to produce recombinant active enterokinase. In this study, the coding sequence of bovine enteropeptidase light chain (bEKL) was isolated from Iranian Sarabi cattle and its expression was investigated in the periplasm and cytoplasm of E. coli by two different expression vectors, pET22 and pET32RH. RNA was extracted from the duodenum part of cattle, cDNA was amplified, the enterokinase light chain coding fragment was cloned and the expression was examined by SDS-PAGE analysis. The higher amounts of soluble enterokinase as a fusion with thioredoxin (Trx) were detected in cytoplasmic expression. The functional enterokinase was purified with a yield of 45 mg per litter by two-steps Ni2+ affinity chromatography. The effective activity of the enzyme implies that it can be produced in large scale for biotechnological applications.