Figure 7
In vitro cleavage of intact fusion proteins by HCV NS3 protease. (A) SDS-PAGE analysis of in vitro cleavage by NS3 protease. Lane M: Protein size marker, Lane 1 & 2: Intact fusion proteins IFNα2b-NS3-LAP and LAP-NS3-IFNα2b respectively. Lane 3 & 4: Cleavage of intact fusion proteins IFNα2b-NS3-LAP and LAP-NS3-IFNα2b respectively by recombinant HCV NS3 protease (in 10:1 molar ratio). Two bands of size 28.5 kDa and 18.1 kDa indicating the LAP fragment and IFNα2b fragment respectively after cleavage of intact fusion proteins (43.5 kDa). (B) Western blot analysis of IFNα2b and LAP fragment produced after cleavage of fusion proteins IFNα2b-NS3-LAP (Lane 1) and LAP-NS3-IFNα2b (Lane 2) by HCV NS3 protease using mouse anti-human IFNα2 antibodies and mouse anti-LAP antibodies as primary antibodies. (C) Standard curve of intact fusion protein plotted against absorbance versus intact fusion protein concentration prepared after sandwich ELISA. (D) Analysis of in vitro cleavage of fusion proteins at different molar ratio of enzyme: fusion protein by sandwich ELISA. Complete cleavage was observed in 1:10 NS3 protease to substrate molar ratio under reaction conditions.
