Role of mouse VH10 and VL gene segments in the specific binding of antibody to Z-DNA, analyzed with recombinant single chain Fv molecules.
A plasmid vector was constructed for the expression of a single chain Fv domain of mouse mAb to Z-DNA (antibody Z22), which is encoded by VH10 and V kappa 10 gene family members along with Dsp2, JH4, and J kappa 4 segments. The vector coded for a PhoA secretion signal, VH segment, flexible peptide linker, VL segment, (His)5, and a protein A domain. Unique restriction sites allowed exchange of the segments as cassettes. Bacteria transformed with the vector secreted soluble recombinant Fv with specific Z-DNA-binding activity. When the L chain of Z22 was replaced with a library of splenic VL cDNA from a mouse immunized with Z-DNA, only a light chain closely resembling that of the original Z22 (differing at six amino acid positions) yielded Fv with Z-DNA-binding activity. The Fv with this L chain replacement had a lowered affinity, but remained selective for Z-DNA. Replacement of the Z22 H chain with a mixture of 11 VH10-encoded H chains yielded two Z-DNA binding clones, but they bound B-DNA and denatured DNA as well as Z-DNA. The replacement clones indicate the importance of the H chain CDR3 and particular VH-VL combinations in formation of specific antibodies to Z-DNA.
Brigido
M M
MM
Department of Biochemistry, Tufts University School of Medicine, Boston, MA 02111.
Polymenis
M
M
Stollar
B D
BD
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology*; D001483:Base Sequence; D004247:DNA / Q000276:immunology*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005803:Genes, Immunoglobulin; D005822:Genetic Vectors; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D007147:Immunoglobulin Light Chains / Q000235:genetics*; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000378:metabolism
D000906:Antibodies; D004268:DNA-Binding Proteins; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins; D004247:DNA