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 heavy chain variable domains joined through a polypeptide linker. A single-chain autoantibody, containing the 14-amino acid 212-polypeptide linker (GSTSGSGKSSEGKG), was constructed based on the light and heavy chain variable region gene sequences of anti-single-stranded DNA autoantibody BV04-01 (IgG2b, kappa). Following protein expression in Escherichia coli, denaturation, refolding, and affinity purification, single-chain autoantibody 04-01 binding with single-stranded DNA and poly(dT) was characterized in solid-phase and solution-phase assays. Homopolymer ligand binding results demonstrated that single-chain autoantibody 04-01 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 thymidine of a (dT)3 ligand.
Department of Microbiology, University of Illinois, Urbana 61801.
C A
CA
Rumbley
L K
LK
Denzin
L
L
Yantz
S Y
SY
Tetin
E W
EW
Voss
D000595:Amino Acid Sequence; D000818:Animals; D000974:Antibodies, Antinuclear / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D001483:Base Sequence; D003001:Cloning, Molecular; D004277:DNA, Single-Stranded / Q000276:immunology* / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011817:Rabbits; D014364:Tryptophan / Q000378:metabolism; D014961:X-Ray Diffraction
D000974:Antibodies, Antinuclear; D004277:DNA, Single-Stranded; D014364:Tryptophan