title: Improving antigenicity of the recombinant hepatitis C virus core protein via random mutagenesis.
abstract: 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(84)S, P(110)S and V(129)L) 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.
authors: Department of Biological Science and Technology, National Chiao Tung University, Hsinchu 30010, Taiwan.
authors: Chen-Ji
authors: CJ
authors: Huang
authors: Hwei-Ling
authors: HL
authors: Peng
authors: Chih-Yu
authors: CY
authors: Cheng
mesh_terms: D000595:Amino Acid Sequence; D000941:Antigens; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D006526:Hepatitis C / Q000175:diagnosis*; D018937:Hepatitis C Antibodies / Q000032:analysis*; D018936:Hepatitis C Antigens / Q000235:genetics* / Q000276:immunology*; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D014758:Viral Core Proteins / Q000235:genetics* / Q000276:immunology*
chemical_list: D000941:Antigens; D018937:Hepatitis C Antibodies; D018936:Hepatitis C Antigens; D011994:Recombinant Proteins; D014758:Viral Core Proteins; C069130:nucleocapsid protein, Hepatitis C virus