Study On The In Vitro Refolding Of Recombinant Bovine Prethrombin-2 Inclusion Bodies

Thrombin is one of the most widely used hemostats. Prethrombin-2 (pThr-2), the smallest single-chain precursor of thrombin, may be activated to thrombin by snake venom. Thrombin is mostly isolated from plasma of animals. pThr-2 gene was cloned to E. coli to produce recombinant prethrombin-2, which was formed as inclusion bodies and must be refolded to obtain native conformation. After pThr-2 inclusion bodies were produced efficiently in the optimized culture conditions, we studied the refolding of pThr-2 in direct dilution process, and further with the assistant of Poly(N-isopropyl acrylamide)(PNIPA) hydrogel and disulfide binding protein A(DsbA).Vector with gene of pThr-2 was successfully transformed into E. coli BL21 (DE3). After the cells were induced by IPTG and further cultured, pThr-2 inclusion bodies were obtained. The fermentation conditions, such as culture medium, IPTG concentration, fermentation temperature, and shake speed for the pThr-2 production were optimized. The inclusion bodies of the pThr-2 produced in the E. coli culture achieved 110 mg/L, accounting for 21.7% of the total cell proteins.After harvested by centrifuge and washed by Tris-HCl buffer(consisting of EDTA, urea and Triton X-100) and solubilized by 8M urea and 0.3M DTT, the key parameters in the refolding process, such as GSSG concentration, GSSG/GSH, urea concentration, L-Arg concentration and operation temperature were investigated by linear regression orthogonal experiments combined with steepest ascent experiments. Good results were obtained under optimized conditions. The total activity of thrombin assayed using chromogenic and fluorogenic substrate was about 2948U/mL at optimum refolding process.It was observed that PNIPA and DsbA were quite efficient in assisting the renaturation of bovine pThr-2. When mixing with 105mg/mL PNIPA hydrogel during the refolding, the thrombin activity was 6222U/mL, which was much higher than that refolded by direct dilution, but the time required for the refolding with PNIPA was a little longer than that by dilution. pAVD63 vector with gene of DsbA was successfully transformed into E. coli to produce DsbA in optimized culture conditions, which was purified by ion-exchange chromatography and used to assist the refolding of pThr-2. When the concentration ratio of DsbA to protein was 4 : 1, the gained thrombin activity was 41.7% higher than that by simple dilution.The thrombin was purified by heparin affinity chromatography for fluorescence spectrum experiments, which indicated the thrombin activated from the refolded pThr-2 had the same emission wavelength with native thrombin.