title: Improving in vivo folding and stability of a single-chain Fv antibody fragment by loop grafting.
abstract: The complementary determining regions (CDRs) from the fluorescein-binding antibody 4-4-20, which yields almost no soluble protein in periplasmic expression in Escherichia coli, were transplanted to the framework of the humanized antibody 4D5. The resulting single-chain Fv fragment (scFv) 4D5Flu showed both a dramatic improvement in soluble expression, even at 37 degrees C, and an improved thermodynamic stability. Antigen affinity was maintained upon this engineering by paying attention to crucial framework-CDR contacts. This demonstrates that the use of superior frameworks is a robust strategy to improve the physical properties of scFv fragments. We also report that the grafted version was selected in phage display over several competing variants of the same antibody with identical binding constant but poorer folding or stability properties. The selection required four panning rounds and a temperature of 37 degrees C and we show that the underlying reason for this selection is a higher fraction of phages carrying functional scFv molecules.
authors.affiliation: Biochemisches Institut der Universität Zürich, Switzerland.
authors.forename: S
authors.initials: S
authors.lastname: Jung
authors.forename: A
authors.initials: A
authors.lastname: Plückthun
mesh_terms: D000595:Amino Acid Sequence; D017104:Bacteriophage M13 / Q000235:genetics; D015153:Blotting, Western; D003001:Cloning, Molecular; D015246:Deoxyribonuclease EcoRI / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D019793:Fluorescein / Q000378:metabolism; D006801:Humans; D007128:Immunoglobulin Fragments / Q000737:chemistry* / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019151:Peptide Library; D011489:Protein Denaturation; D015202:Protein Engineering; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification; D017421:Sequence Analysis
chemical_list: D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D011994:Recombinant Proteins; C055966:immunoglobulin Fv; D015246:Deoxyribonuclease EcoRI; D019793:Fluorescein