Clone,expression,purification and activity characterization of Jian carp(Cyprinus carpio var. Jian) Cathepsin L

CAT L gene fragment which encoded mature peptide of Jian carp was cloned using TA clone and characterized by double enzyme cutting firstly,prokaryotic expression vector CAT L-p ET-30 a was constructed and transformed into E.coli BL21 subsequently. Recombinant CAT L was expressed after induced by 1 mmol/L IPTG at 37 ℃ for 2 h. And the objective protein was gradiently washed by urea and purified by Ni2 +-NTA agarose affinity chromatography,SDS-PAGE was conducted to examine the results of expression and purification. Activity assay(Z-Phe-Arg-MCA as a substrate) was finally used to characterize the p H and thermal stability of CAT L,and illustrated the effects of common additives used in surimi product and frozen storage on its activity stability. The results of double enzyme cutting indicated that CAT L gene fragment was successfully cloned,and shared 99.11% sequence identities with the CAT L of common carp. The analysis of SDS-PAGE illustrated that the recombinant CAT L with the molecular weight of 28 ku was highly purified.Activity assay revealed that CAT L was stable ranging from 20 ℃ to 50 ℃ and p H 3.0 to 6.5,the activity of CAT L was inhibited by Na Cl and Na4P2O7 and appeared dose-dependence,while sucrose and sorbitol had no effects on the activity of CAT L. CAT L of Jian carp was successfully cloned,expressed and purified,and effects of heat,p H,additives on the activity stability of CAT L was illustrated.