title: Selecting for antibody scFv fragments with improved stability using phage display with denaturation under reducing conditions.
abstract: Stability of single-chain Fvs (scFvs) can be improved by mutagenesis followed by phage display selection where the unstable variants are first inactivated by, for example, denaturing treatment. Here we describe a modified strategy for the selection of stabilized antibody fragments by phage display, based on denaturation under reducing conditions. This strategy was applied to an anti-thyroid-stimulating hormone (TSH) scFv fragment which refolded remarkably during the selection if denaturation was carried out in conventionally used non-reducing conditions. Refolding was, however, efficiently prevented by combining denaturation with reduction of the intra-domain disulfide bridges, which created favourable conditions for selection of clones with improved stability. Using this strategy, scFv mutants with 8-9 degrees C improved thermal stability and 0.8-0.9 M improved stability for guanidinium chloride were found after 4-5 enrichment cycles. The most stable mutants selected contained either Lys(H)66Arg or Asn(H)52aSer mutations, which are known to stabilize other scFvs. Periplasmic expression level of the mutants was also improved.
authors.affiliation: Department of Biotechnology, University of Turku, Tykistökatu 6A 6th floor, FIN-20520 Turku, Finland. eechbr@utu.fi
authors.forename: Eeva-Christine
authors.initials: EC
authors.lastname: Brockmann
authors.forename: Matthew
authors.initials: M
authors.lastname: Cooper
authors.forename: Nelli
authors.initials: N
authors.lastname: Strömsten
authors.forename: Markus
authors.initials: M
authors.lastname: Vehniäinen
authors.forename: Petri
authors.initials: P
authors.lastname: Saviranta
mesh_terms: D000818:Animals; D004220:Disulfides / Q000737:chemistry; D019791:Guanidine / Q000737:chemistry; D006358:Hot Temperature; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D018828:Immunoglobulins, Thyroid-Stimulating / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D051379:Mice; D016296:Mutagenesis / Q000235:genetics; D010084:Oxidation-Reduction; D019151:Peptide Library; D017354:Point Mutation / Q000235:genetics; D011489:Protein Denaturation / Q000235:genetics; D017510:Protein Folding; D013816:Thermodynamics
chemical_list: D004220:Disulfides; D007135:Immunoglobulin Variable Region; D018828:Immunoglobulins, Thyroid-Stimulating; D019151:Peptide Library; D019791:Guanidine