Tandem affinity tags for the purification of bivalent anti-DNA single-chain Fv expressed in Escherichia coli.
Antibodies to DNA define an important autospecificity that arises in systemic lupus erythematosus (SLE). To elucidate the molecular features that may explain the pathogenesis of SLE, a heterologous system for expression of cloned V genes is often desirable. Here, a single-chain Fv coding domain was constructed by using the heavy- and light-chain V genes of a high-affinity site-directed mutant of the murine anti-dsDNA autoantibody, 3H9. This scFv was joined in frame to the c-jun leucine zipper for dimerization, and to two affinity tags, domain B of the staphylococcal protein A and a pentahistidine peptide, for purification. Dimerization of the scFv was determined by size-exclusion chromatography. The yields of the scFv following affinity purification on IgG agarose or Ni-NTA agarose were compared, and the activities of the resulting protein fractions were determined. A two-step purification of periplasmic extracts on Ni-NTA agarose and IgG agarose, followed by elution with 3.5 M MgCl(2), yielded scFv with the highest specific activity. The final purified material bound DNA by ELISA, electrophoretic mobility shift assay, and immunofluorescence of fixed Hep-2 cells. Antibodies purified in this fashion should have applications in structure/function studies in which it is essential to generate highly purified antigen-combining sites.
Cocca
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BA
Department of Microbiology and Immunology, MCP Hahnemann University, Philadelphia, Pennsylvania, 19129, USA.
Seal
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Radic
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D000345:Affinity Labels / Q000378:metabolism*; D000818:Animals; D000974:Antibodies, Antinuclear / Q000302:isolation & purification*; D000918:Antibody Specificity; D000937:Antigen-Antibody Reactions; D001323:Autoantibodies; D001483:Base Sequence; D002846:Chromatography, Affinity / Q000379:methods; D004247:DNA / Q000276:immunology*; D004268:DNA-Binding Proteins; D019281:Dimerization; D017886:Durapatite; D004926:Escherichia coli / Q000276:immunology; D015870:Gene Expression; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry* / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000302:isolation & purification*; D016350:Leucine Zippers; D051379:Mice; D009532:Nickel; D009571:Nitrilotriacetic Acid / Q000031:analogs & derivatives; D009942:Organometallic Compounds; D019897:Periplasm; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012685:Sepharose
D000345:Affinity Labels; D000974:Antibodies, Antinuclear; D001323:Autoantibodies; D004268:DNA-Binding Proteins; D007074:Immunoglobulin G; D007135:Immunoglobulin Variable Region; D009942:Organometallic Compounds; D011994:Recombinant Proteins; C088321:nickel nitrilotriacetic acid; D009532:Nickel; D004247:DNA; D012685:Sepharose; D017886:Durapatite; D009571:Nitrilotriacetic Acid