Fig. 1

Cloning strategy, expression, and purification of NS3-NS4A of HCV genotype 3a. A The codon-optimized nucleotide sequence encoding NS3 protease domain fused to the NS4A cofactor was cloned in the pET11a plasmid under the control of the T7 promoter using NdeI and BamHI restriction sites. The construct was used for heterologous expression of NS3-NS4A fused to a polyhistidine (His₇) tag in E. coli. B The soluble form of NS3-NS4A expressed in E. coli BL21 (DE3) cells was analyzed through SDS-PAGE. Lane 1 represents the soluble fraction of the cell lysate (from the cells induced with IPTG) analyzed on a 4–12% Bis–Tris NuPAGE gel. The protein in the native form was successfully purified through affinity chromatography and gel filtration. Lane 2 shows the sample collected from the HisTrap column during the elution step, lanes 3 and 4 depict the digestion mixture (containing His-tagged AcTEV protease and NS3-NS4A), lane 5 represents the sample collected during reverse affinity chromatography, and lane 6 shows the sample collected after gel filtration. Lane M represents the mobility of proteins with known molecular weights (SeeBlue Pre-stained Protein Marker)