Improving antigenicity of the recombinant hepatitis C virus core protein via random mutagenesis

In order to enhance the sensitivity of diagnosis, a recombinant clone containing domain I of HCV core (amino acid residues 1 to 123) was subjected to random mutagenesis. Five mutants with higher sensitivity were obtained by colony screening of 616 mutants using reverse ELISA. Sequence analysis of these mutants revealed alterations focusing on W ⁸⁴, P ⁹⁵, P ¹¹⁰, or V ¹²⁹. The inclusion bodies of these recombinant proteins overexpressed in E. coli BL21(DE3) were subsequently dissolved using 6 M urea and then refolded by stepwise dialysis. Compared to the unfolded wild-type antigen, the refolded M3b antigen (W ⁸⁴S, P ¹¹⁰S and V ¹²⁹L) exhibited an increase of 66% antigenicity with binding capacity of 0.96 and affinity of 113 μM ^-1. Moreover, the 33% decrease of the production demand suggests that M3b is a potential substitute for anti-HCV antibody detection.