The Study Of Enterokinase Produced By Recombinant E.coli

Enterokinase(EK) is a serine proteinase which consists of a high chain and a light chain connected by a disulfide bond. The light chain consists of a chymotrypsin-like proteinase domain and is sufficient for the normal recognition of EK substrate. EK allows any downstream fusion target protein to retain its native amino-terminus, without leaving any unwanted amino acid residues on their amino termini. So now it is used very often in pharmacy industry. Currently most commercial Eks are holoenzymes purfied from bovine or porcine intestines, and are expensive. Even highly puried preparations are prone to be contaminated by traces of other gut protease.In this paper, two recombinant E. coli strains (BL21(DE3)/ pET39-sBEKLClKX and BL21(DE3)/ pET39-sBEKLClSX) were constructed with two expression plasmids including T₇lac promoter, (His) 6-tag and enterokinase gene. The strains were cultured in LB medium containing 50ug/mL kanamycin at 37℃. When cell density (OD₆₀₀) reached 0.5-0.6, IPTG was added. When BL21(DE3)/ pET39-sBEKLClSX was induced with 0.1mM IPTG for 8 hours at 26℃, the highest sBEKLC's activity was obtained;as for BL21(DE3)/ pET39-sBEKLClKX the suitable induction conditions were: 26℃, 0.2mM IPTG for 8 hours. Then sBEKLC was separated by cold osmotic shock technique, and further purified by nickel affinity chromatography. The results indicated that the purity of enterokinase was higher than 95%, and 5ng sBEKLC1SX could cleave 20ug TrxA-HBD4 effectively after 16h reaction.The fusion protein was mainly expressed in the form of inclusion bodies. In order to refold the inclusion bodies, we used affinity chromatography to refold the protein according to the his-tag of the target protein. The cells were collected and disrupted, and the inclusion bodies were collected by centrifugation. After washing, the inclusion bodies were dissolved in a 8M urea solution(pH8.0) containing 100 mM GSH and 50mM Tris. The dissolved protein was further purified and refolded by Ni²⁺ affinity column to obtain active enterokinase. The results indicated that the purity of enterokinase was higher than 96%, and the refolded sBEKLC cleaved TrxA-HBD4 after 30h reaction with a high rate (71.2%).