Investigation of enterokinase light chain cloning and expression in Escherichia coli

Background and Aim: Enterokinase, a serine protease which consists of a heavy chain and a light chain. The light chain of enterokinase is the catalytic subunit; that recognizes DDDDKX site. Enterokinase light chain has ۴ disulfide bonds. At the presence of the DsbA (Disulfide bond) sequence in the design of the enterokinase gene, the protein is transferred to the periplasmic space, in which the disulfide bond is formed and the protein folded correctly. Enterokinase allows any recombinant protein to retain its native N-terminus without leaving any unwanted residues on their amine end. Due to the importance of production of enterokinase light chain in industry, we cloned the enterokinase light chain gene and investigated different expression conditions of the protein to achieve an optimized condition for expressing the protein.Methods: The nucleotide sequence of the enterokinase gene was obtained for expression in the bacteria. The sequence was synthesized in pUC۵۷ vector. In the synthesized construct, DsbA tag and then gene is located between the Nde I and Xho I restriction sites. Then, it was cloned into pET-۲۱a+ and the vector was transformed to the E. coli. The cloning was confirmed with sequencing. The expression of enterokinase was investigated in different concentrations of Isopropyl β-D-۱-thiogalactopyranoside (IPTG) and lactose and different conditions of temperature and time with appropriate aeration in order to find optimal conditions for expression. Cold osmotic shock method was used to isolate enterokinase from periplasmic space. Expression analysis was performed by Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and it was observed that more protein was soluble in the periplasmic space.Results: The constructed expression vectors, pET۲۱-enterokinase light chain, were transformed into E. coli BL۲۱. To observe the expression level of enterokinase, LB medium was used to cultivate this recombinant E. coli. According to SDS-PAGE analysis, the enterokinase was successfully expressed under the optimized condition (i.e. ۲ mM of lactose induction at a cell density of OD۶۰۰ ¼ ۱ at ۳۷°C and a post-induction time of ۱۸ hours).Conclusion: The enterokinase light chain gene was successfully transformed and its expression was optimized within the E. coli.

کلیدواژه ها:

Enterokinase ، catalytic subunit ، periplasmic space ، Cold osmotic shock