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.

Figure 1

Sequence comparison. (a) The primary sequence of wild-type recombinant HCV core antigen presented in single letter code. It consists of three components: pelB leader (from aa 1 to aa 31 in border-box), HCV core fragment containing domain I (from aa 32 to aa 154, total 123 aa), and His tag (from aa 155 to aa 162, underlined). (b) Five mutants with higher antigenicities are aligned with wild-type antigen covering residues 68 to 137. Four high-frequency mutation points at W⁸⁴, P⁹⁵, P¹¹⁰, and V¹²⁹ are in boldface. The mutations are marked with a shaded box. The characters H, E, and C stand for alpha helices, beta strands, and random coil, respectively.

Figure 2

HCV ELISA for urea-denatured HCV core mutants. The urea-denatured HCV core antigens (100 μL of 4 μg/mL per well) were used to coat the microplate for the detection of anti-HCV antibody of BBI panel (a) and GBC inhouse panel (b). The absorbance data were compiled statistically as S/Co value (S: sample value; Co: cut-off value). The cut-off value was calculated as the negative control OD plus positive control OD divided by 4 (Co = NCx + PCx/4). The positive samples (i.e., S/Co > 1, marked as open circle, lot number HCV10165-6∼9) could be clearly separated from the negative samples (i.e., S/Co < 1, marked as closed circle, lot number HCV10017-1∼7, HCV10026-1∼5, HCV10058-1∼3) of BBI panel. In Figure 2(b), the primary antibodies used are sequentially diluted in 10-fold order from 10x to 70x of the PC (positive control) and NC (negative control). Co value was calculated by the average OD values of NC and 20x PC. The data are marked as • (10x), ∘ (20x), ▾ (30x), ▽(40x), ■ (50x), □ (60x), ♦ (70x), and (negative control).

Figure 3

Saturation curves for urea-denatured (a) and refolded (b) forms of HCV core antigens. Antigenicity of unfolded (a) and refolded (b) forms of HCV core antigens were examined by ELISA using the microplate that had been coated with different amounts of Wt (•), M1a (∘), M1b (▾), M2 (▽), M3a (■), and M3b(□).