Expression, Refolding, and Ferritin-Binding Activity of the Isolated VL-Domain of Monoclonal Antibody F11

A. P. Dubnovitsky¹, Z. I. Kravchuk¹, A. A. Chumanevich¹, A. Cozzi², P. Arosio², and S. P. Martsev^1*

¹Institute of Bioorganic Chemistry, National Academy of Sciences of Belarus, ul. Akademika Kuprevicha 5/2, Minsk, 220141 Belarus; fax: +375-17-2-63-7274; E-mail: martsev@ns.iboch.ac.by

²DIBIT, Scientific Institute H. San Raffaele, Milano, Italy

^* To whom correspondence should be addressed.

Received April 14, 2000

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Expression of the VL-domain of mouse monoclonal antibody F11 to human spleen ferritin in Escherichia coli cells is associated with the formation of insoluble protein aggregates (inclusion bodies). The aggregates were solubilized in the presence of guanidine hydrochloride and the recombinant VL-domain was purified by immobilized metal affinity chromatography (IMAC). Subsequent renaturation results in ~99% pure preparation with high yield. The VL-domain forms dimers at concentrations from 1 to 10 mg/ml. Monomeric form is detected only at protein concentrations below 0.5 mg/ml. Functional activity of the VL-domain was verified by two variants of ELISA. The affinity of the VL-domain ((0.2-1.2)·10⁸ M^-1) is similar to the affinity of the 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-fold lower than that in the case of F11 antibody. In another ELISA system with immobilized ferritin, affinity was decreased 30-fold. The VL-domain of antibody F11 is the first example of the recombinant variable domain of the immunoglobulin light chain that preserves the antigen-binding activity in the absence of the partner VH-domain. The data indicate that the recombinant VL-domain can be used in construction of chimeric immunotoxins and other antigen-binding proteins in immunotherapy and in studies of correlations between folding, stability, and activity of immunoglobulins.

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KEY WORDS: VL-domain, recombinant antibody, immunoglobulin domain, folding, protein expression, antigen binding

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