The Of Hasnpv Chitinase Gene Prokaryotic Expression, Purification And Refolding

Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HaSNPV) had been extensively used for the control of H. armigera since the first isolation of this pathogen in Hubei province of China. The genome of this virus has been completely sequenced and a chitinase gene belonging to family 18 chitinases was identified. The chitinase was found to be essential in liquefying infected cadavers.Chitinase gene from HaSNPV was constructed into expression vector pET28a(+) and then transferred into expression host Rosetta(DE3), which was designed to enhance expression of eukaryotic proteins that contained codons rarely used in E. coli. The chitinase gene was successfully expressed as a fusion protein (His)6- chiA with a His-tag at its N terminal. After the recombinant E.coli was induced with 0.5 mM IPTG for 3h, the percentage of the recombinant protein could get to 22.6% of the total E.coli protein, and 1mL culture medium could produce 65μg (His)6- chiA protein. But it was mainly expressed as inclusion body and had no activity, so we constructed the gene into another expression vector pMAL- c2X. About 14.7% of the total E.coli protein was MBP- chiA, and 30% of MBP- chiA protein was soluble. But this fusion protein still had no activity.The inclusion body was expressed in Rosetta(DE3) which contained pET28a(+)-chiA plasmid . (His)6- chiA protein was then purified by Ni-NTA column affinity chromatography under denaturing conditions (8M urea). The (His)6- chiA protein was then refolded by stepwise dialysis with the urea concentration in the base dialysis buffer reduced as follows 6M-5M-4M-3M-2M-1M-0M . After stepwise dialysis, about 28% protein was refolded, and the enzymatic activation of 1 mg refolded (His)6-chiA was 0.3U . The (His)6- chiA protein was also successfully refolded while it was immobilized on Ni-NTA, by washing it using refolding buffer with urea concentration as follows 6M-5M-4M-3M-2M-1M-0M. After that about 43% protein was refolded, and the enzymatic activation of 1 mg refolded protein could get to 0.5U.