Bacterial expression of anti-DNA antibody domains.
Bacterial production of recombinant Fab or Fv domains of antibodies is an important tool for analyzing structural correlates of antigen binding or idiotype expression. Bacterial products may be Fab or Fv molecules that assemble from separate chains in the periplasm or a single-chain Fv protein. This article describes properties and applications of a plasmid vector used for production of single-chain Fv (scFv). The expression cassette, initially designed for production of the Fv domain of anti-Z-DNA mAb Z22, has a bacterial secretion signal that permits secretion of soluble scFv into growth medium. A single B domain of staphylococcal protein A facilitates affinity purification and generic assay of products independent of antigen-binding activity. Gene segment swapping and directed mutagenesis identified structural features important for Z-DNA binding and revealed the structural similarity of autoantibody and immunization-induced antibody. The H and L regions of mAb Z22 are readily replaced by those of any cloned Ig, a library of V regions, or other proteins. Modified forms of the vector code for production of separate H or L chain V regions, which can associate with each other to form functional Fv complexes. Although there is large variation in the yield with different V regions, most clones provide enough soluble product for antigen-binding assays. Current developments are aimed at increasing the yield consistency to allow production of enough material for three-dimensional structural analysis.
Stollar
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BD
Department of Biochemistry, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.
D000974:Antibodies, Antinuclear / Q000096:biosynthesis* / Q000737:chemistry / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D001419:Bacteria / Q000378:metabolism*; D015153:Blotting, Western; D003001:Cloning, Molecular; D004247:DNA / Q000276:immunology / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics*; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology*
D000974:Antibodies, Antinuclear; D000911:Antibodies, Monoclonal; D004268:DNA-Binding Proteins; D007140:Immunoglobulin Fab Fragments; D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins; D004247:DNA