Ca. Rumbley et al., CONSTRUCTION, CHARACTERIZATION, AND SELECTED SITE-SPECIFIC MUTAGENESIS OF AN ANTI-SINGLE-STRANDED DNA SINGLE-CHAIN AUTOANTIBODY, The Journal of biological chemistry, 268(18), 1993, pp. 3667-3674
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.