关键词: fibrolase, 大肠杆菌, 表达, 复性

Abstract: Fibrolase is a non-hemorrhagic zinc metalloproteinase isolated from southern copperhead snake venom (Agkistrodon contortrix contortrix) and capable of degrading fibrin clots through specific cleavage of the Aα-chains of fibrin(ogen) at the position Lys413?Leu414. The DNA encoding of fibrolase was amplified by PCR and cloned into the pET25b(+) vectors and high-level expression in E. coli BL21(DE3) as inclusion bodies, the recombinant fibrolase was about 40% of the total cell proteins. Compared with the simple batch dilution refolding method, fed-batch dilution refolding and dialysis refolding methods are more efficient in this work for fibrolase refolding. At the same time, the experimental conditions, including temperature, pH, denatured protein concentration, GSSG concentration and GdmCl concentration in the renaturation process of recombinant fibrolase inclusion body were studied. Good results are obtained when refolding of fibrolase was carried out by fed-batch dilution refolding method at pH 8.5, 4℃, and refolding buffer consisted of 1.5 mol/L GdmCl, 1 mmo1/L GSSG and 2 mmo1/L GSH, and sample concentration is 10 mg/mL, dilution factor is 50. The recovery rate of recombinant fibrolase was about 25% after refolding and purification.

Key words: fibrolase, E. coli, expression, refolding