Expression, refolding, and ferritin-binding activity of the isolated VL-domain of monoclonal antibody F11

Expression of the VL-domain of mouse monoclonal antibody F11 to human splee n ferritin in Escherichia coli cells is associated with the formation of in soluble protein aggregates (inclusion bodies). The aggregates were solubili zed in the presence of guanidine hydrochloride and the recombinant VL-domai n was purified by immobilized metal affinity chromatography (IMAC). Subsequ ent renaturation results in similar to 99% pure preparation with high yield . The VL-domain forms dimers at concentrations from 1 to 10 mg/ml. Monomeri c form is detected only at protein concentrations below 0.5 mg/ml. Function al activity of the VL-domain was verified by two variants of ELISA The affi nity of the VL-domain ((0.2-1.2).10(8) M-1) is similar to the affinity of t he full-length parental antibody F11 because when the immobilized VL-domain was used, the binding constant of ferritin to the VL-domain was only 4-6-f old lower than that in the case of F11 antibody. In another ELISA system wi th immobilized ferritin, affinity was decreased 30-fold. The VL-domain of a ntibody F11 is the first example of the recombinant variable domain of the immunoglobulin light chain that preserves the antigen-binding activity in t he absence of the partner VH-domain. The data indicate that the recombinant VL-domain can be used in construction of chimeric immunotoxins and other a ntigen-binding proteins in immunotherapy and in studies of correlations bet ween folding, stability, and activity of immunoglobulins.