Fig. 4

Construction, purification, and renaturation of MBP-C13scFv-His. a Amino acid sequences of MBP-C13scFv-His. The region of 413–677 corresponds to THETAL (see below). b Purification of MBP-C13scFv-His. Lane 1, insoluble fraction obtained after sonication of E. coli BL21(DE3) overexpressing MBP-C13scFv-His; lane 2, insoluble fraction after washing with detergents; lane 3, urea-extracted fraction; lane 4, residual pellet after extraction by urea; lane 5, refolded MBP-C13scFv-His. c Binding activities of MBP-C13scFv-His refolded at various pH. MBP-C13scFv-His was refolded by using refolding buffer adjusted to different pH. After dialysis against PBS (pH 7.4), the binding activities to cCRY4CCE were measured by ELISA using anti-6xHis antibody (Wako), changing the concentration of MBP-C13scFv-His. The half-maximal effective concentrations (EC₅₀) of the immunoreactions were calculated by fitting the value to the Rodbard model using ImageJ. The numbers of observations are three