Construction and characterization of a quadruplex DNA selective single-chain autoantibody from a viable motheaten mouse hybridoma with homology to telomeric DNA binding proteins.
An autoantibody produced by a hybridoma derived from a viable motheaten mouse was isolated and found to have moderately high binding affinity for nucleic acids and specific preference for quadruplex DNAs. Polymerase chain reaction primers were designed to link the cloned parental antibody variable region fragments together in a subcloning vector. This single-chain variable fragment construct was then subcloned into the T7 promoter-driven expression vector pET22b(+). The construct contains (N- to C-terminal) a pelB leader sequence, variable heavy chain, glycine-serine polylinker, variable light chain, and biotin mimic peptide "strep-tag" sequence (pelB-VH-linker-VL-strep-tag). The ca. 29 kDa protein was expressed, exported to the periplasmic space of NovaBlue (DE) Escherichia coli, and purified by streptavidin affinity chromatography by binding the fused strep-tag peptide. The specificity of the purified single-chain variable fragment (scFv) for quadruplex and duplex DNAs was evaluated by a radioimmunofilterbinding assay. It retained about 10-fold higher affinity for quadruplexes relative to duplex DNA, a reduction of ca. 4-fold from the relative preferences of the parent IgG. The complementary-determining regions contain sequences that are homologous to or conservatively divergent from the key DNA-binding helix-turn-helix-forming motifs of Myb/RAP1 family telomeric DNA-binding proteins (1-3). The presence of this antibody in the autoimmune repertoire suggests a possible linkage between autoimmunity, telomeric DNA binding proteins, and aging.
Brown
J C
JC
Department of Biochemistry, North Carolina State University, Raleigh 27695, USA.
Brown
B A
BA
Li
Y
Y
Hardin
C C
CC
D000595:Amino Acid Sequence; D000818:Animals; D000974:Antibodies, Antinuclear / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000276:immunology* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry*; D054856:G-Quadruplexes; D006825:Hybridomas / Q000737:chemistry*; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D051379:Mice; D008810:Mice, Inbred C57BL; D008817:Mice, Mutant Strains; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D015202:Protein Engineering / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid; D057127:Single-Chain Antibodies; D016615:Telomere / Q000378:metabolism
D000974:Antibodies, Antinuclear; D004268:DNA-Binding Proteins; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies; C115959:anti-quadruplex DNA scFv; D004247:DNA