title: Expression and purification of a single-chain variable fragment antibody derived from a polyol-responsive monoclonal antibody.
abstract: A previously described polyol-responsive monoclonal antibody (PR-mAb) was converted to a single-chain variable fragment (scFv). This antibody, PR-mAb NT73, reacts with the beta' subunit of Escherichia coli RNA polymerase and has been used for the immunoaffinity purification of polymerase. mRNAs encoding the variable regions of the heavy chain (VH) and light chain (VL) were used as the template for cDNA synthesis. The sequences were joined by the addition of a "linker" sequence and then cloned into several expression vectors. A variety of expression plasmids and E. coli hosts were used to determine the optimal expression system. Expression was highest with the pET22b(+) vector and the Rosetta(DE3)pLysS host strain, which produced approximately 60 mg purified His-tagged scFv per liter of culture (3.3 g wet weight cells). Although the production of soluble scFv was preferred, overproduced scFv formed inclusion bodies under every expression condition. Therefore, inclusion bodies had to be isolated, washed, solubilized, and refolded. The FoldIt protein refolding kit and enzyme-linked immunosorbent assay were sequentially used to determine the optimal refolding conditions that would produce active His-tagged scFv. Immobilized metal affinity chromatography was used for the final purification of the refolded active scFv. The polyol-responsiveness of the scFv was determined by an ELISA-elution assay. Although the scFv loses considerable affinity for its antigen, it maintains similar polyol-responsiveness as the parent monoclonal antibody, PR-mAb NT73.
authors.affiliation: McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, 1400 University Avenue, Madison, WI 53706, USA. jalamberski@wisc.edu
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Lamberski
authors.forename: Nancy E
authors.initials: NE
authors.lastname: Thompson
authors.forename: Richard R
authors.initials: RR
authors.lastname: Burgess
mesh_terms: D000595:Amino Acid Sequence; D000665:Ampholyte Mixtures / Q000494:pharmacology; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D001483:Base Sequence; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D007128:Immunoglobulin Fragments / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D011108:Polymers / Q000494:pharmacology; D017510:Protein Folding
chemical_list: D000665:Ampholyte Mixtures; D000911:Antibodies, Monoclonal; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D011108:Polymers; C024617:polyol