title: Biosynthetic antibody binding sites: development of a single-chain Fv model based on antidinitrophenol IgA myeloma MOPC 315.
abstract: The functional antigen binding region of antidinitrophenol mouse IgA myeloma MOPC 315 has been produced as a single-chain Fv (sFv) protein in E. coli. Recombinant 315 proteins included sFv alone, a bifunctional fusion protein with amino-terminal fragment B (FB) of staphylococcal protein A, and a two-chain 315 Fv fragment. Successful refolding of the 315 sFv required formation of disulfide bonds while the polypeptide was in a denatured state, as previously observed for the parent Fv fragment. Affinity-purified recombinant 315 proteins showed full recovery of specific activity, with values for Ka,app of 1.5 to 2.2 x 10(6) M-1, equivalent to the parent 315 Fv fragment. As observed for natural 315 Fv, the sFv region of active FB-sFv315 fusion protein was resistant to pepsin treatment, whereas inactive protein was readily degraded. These experiments will allow the application of protein engineering to the 315 single-chain Fv; such studies can advance structure-function studies of antibody combining sites and lead to an improved understanding of single-chain Fv proteins.
authors.affiliation: Creative BioMolecules, Inc., Hopkinton, Massachusetts 01748.
authors.forename: J E
authors.initials: JE
authors.lastname: McCartney
authors.forename: L
authors.initials: L
authors.lastname: Lederman
authors.forename: E A
authors.initials: EA
authors.lastname: Drier
authors.forename: N A
authors.initials: NA
authors.lastname: Cabral-Denison
authors.forename: G M
authors.initials: GM
authors.lastname: Wu
authors.forename: R S
authors.initials: RS
authors.lastname: Batorsky
authors.forename: J S
authors.initials: JS
authors.lastname: Huston
authors.forename: H
authors.initials: H
authors.lastname: Oppermann
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001666:Binding Sites, Antibody; D004247:DNA / Q000235:genetics; D004140:Dinitrophenols / Q000276:immunology*; D051379:Mice; D008969:Molecular Sequence Data; D009101:Multiple Myeloma / Q000276:immunology*; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000235:genetics
chemical_list: D004140:Dinitrophenols; D011993:Recombinant Fusion Proteins; D004247:DNA