Figure 2

Recombinant forms of collagen VII used in this study. (a) Schematic representation of human collagen VII consisting of a central collagenous domain flanked by a large, 145 kDa N-terminal non-collagenous domain and a smaller, 30 kDa non-collagen domain at its C-terminus. The collagenous domain is interrupted by a 39 amino acid non-collagenous hinge region. The recombinant forms of collagen VII generated in this study are two N-terminally 8xhistidine tagged chimeric proteins termed His-hCVII-NC1-NC2 and His-hCVII-NC1-H-NC2 corresponding to the fused NC1 and NC2 domains (aa 1-1278, 2776-2944) and to the fused NC1, hinge and NC2 regions (1-1278, 1940-1979, 2776-2944) of the antigen, respectively. (b) Sodium dodecylsulfate-polyacrylamide gel electrophoresis of the purified recombinant His-hCVII-NC1-NC2 and His-hCVII-NC1-H-NC2 proteins shows their migration at around 153 (lane 2) and 158 kDa (lane 3), respectively. Weight markers of 200, 116, 97, 66 and 45 kDa are shown in lane 1. (c) Immunoblot analysis of the two recombinant proteins His-hCVII-NC1-NC2 (lanes 1 and 4) and His-hCVII-NC1-H-NC2 (lanes 2 and 5) as well as a recombinant form of collagen XVII [47] (lanes 3 and 6) using a monoclonal antibody specific to the NC1 domain of collagen VII (clone LH7.2; lanes 1-3) and a monoclonal antibody specific to soluble ectodomain of collagen XVII [47] (lanes 4-6).