CONSTRUCTION, CHARACTERIZATION, AND SELECTED SITE-SPECIFIC MUTAGENESIS OF AN ANTI-SINGLE-STRANDED DNA SINGLE-CHAIN AUTOANTIBODY

Single-chain antibodies are comprised of immunoglobulin light and heav y chain variable domains joined through a polypeptide linker. A single -chain autoantibody, containing the 14-amino acid 212-polypeptide link er (GSTSGSGKSSEGKG), was constructed based on the light and heavy chai n variable region gene sequences of anti-single-stranded DNA autoantib ody BV04-01 (IgG2b,kappa). Following protein expression in Escherichia coli, denaturation, refolding, and affinity purification, single-chai n autoantibody 04-01 binding with single-stranded DNA and poly(dT) was characterized in solid-phase and solution-phase assays. Homopolymer l igand binding results demonstrated that single-chain autoantibody 04-0 1 possessed anti-DNA binding properties similar to BV04-01 IgG and Fab fragments. Based on x-ray crystallographic analyses of BV04-01, site- specific mutagenesis studies were conducted on 2 residues (L32Tyr and H100aTrp) involved in aromatic stacking interactions with the middle t hymidine of a (dT)3 ligand.