Construction, characterization, and selected site-specific mutagenesis of an anti-single-stranded DNA single-chain autoantibody

C. A. Rumbley, L. K. Denzin, L. Yantz, S. Y. Tetin, E. W. Voss

Research output: Contribution to journalArticlepeer-review

36 Scopus citations

Abstract

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,κ). 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 (L32(Tyr) and H100a(Trp)) involved in aromatic stacking interactions with the middle thymidine of a (dT)3 ligand.

Original languageEnglish (US)
Pages (from-to)13667-13674
Number of pages8
JournalJournal of Biological Chemistry
Volume268
Issue number18
StatePublished - 1993
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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