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 system ic lupus erythematosus (SLE). To elucidate the molecular features that may explain the pathogenesis of SLE, a heterologous system for expression of cl oned 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 tw o affinity tags, domain B of the staphylococcal protein A and a pentahistid ine peptide, for purification. Dimerization of the scFv was determined by s ize-exclusion chromatography. The yields of the scFv following affinity pur ification on IgG agarose or Ni-NTA agarose were compared, and the activitie s of the resulting protein fractions were determined. A two-step purificati on of periplasmic extracts on Ni-NTA agarose and IgG agarose, followed by e lution with 3.5 M MgCl2, yielded scFv with the highest specific activity. T he final purified material bound DNA by ELISA, electrophoretic mobility shi ft assay, and immunofluorescence of fixed Hep-a cells. Antibodies purified in this fashion should have applications in structure/function studies in w hich it is essential to generate highly purified antigen-combining sites. ( C) 1999 Academic Press.