Tandem affinity tags for the purification of bivalent anti-DNA single-chain Fv expressed in Escherichia coli

Brian A. Cocca, Samarendra N. Seal, Marko Radic

Research output: Contribution to journalArticle

10 Citations (Scopus)

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 MgCl2, 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.

Original languageEnglish (US)
Pages (from-to)290-298
Number of pages9
JournalProtein Expression and Purification
Volume17
Issue number2
DOIs
StatePublished - Jan 1 1999

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Single-Chain Antibodies
Sepharose
Escherichia coli
DNA
Dimerization
Systemic Lupus Erythematosus
Immunoglobulin G
Leucine Zippers
Magnesium Chloride
Antibodies
Staphylococcal Protein A
Electrophoretic Mobility Shift Assay
Autoantibodies
Genes
Gel Chromatography
Fluorescent Antibody Technique
Enzyme-Linked Immunosorbent Assay
Binding Sites
Light
Antigens

All Science Journal Classification (ASJC) codes

  • Biotechnology

Cite this

Tandem affinity tags for the purification of bivalent anti-DNA single-chain Fv expressed in Escherichia coli. / Cocca, Brian A.; Seal, Samarendra N.; Radic, Marko.

In: Protein Expression and Purification, Vol. 17, No. 2, 01.01.1999, p. 290-298.

Research output: Contribution to journalArticle

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