title: Tandem affinity tags for the purification of bivalent anti-DNA single-chain Fv expressed in Escherichia coli.
abstract: 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.
authors.affiliation: Department of Microbiology and Immunology, MCP Hahnemann University, Philadelphia, Pennsylvania, 19129, USA.
authors.forename: B A
authors.initials: BA
authors.lastname: Cocca
authors.forename: S N
authors.initials: SN
authors.lastname: Seal
authors.forename: M Z
authors.initials: MZ
authors.lastname: Radic
mesh_terms: 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
chemical_list: 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