Selecting for antibody scFv fragments with improved stability using phage display with denaturation under reducing conditions.
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.
Brockmann
Eeva-Christine
EC
Department of Biotechnology, University of Turku, Tykistökatu 6A 6th floor, FIN-20520 Turku, Finland. eechbr@utu.fi
Cooper
Matthew
M
Strömsten
Nelli
N
Vehniäinen
Markus
M
Saviranta
Petri
P
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
D004220:Disulfides; D007135:Immunoglobulin Variable Region; D018828:Immunoglobulins, Thyroid-Stimulating; D019151:Peptide Library; D019791:Guanidine