title: Ribosome display efficiently selects and evolves high-affinity antibodies in vitro from immune libraries.
abstract: Ribosome display was applied for affinity selection of antibody single-chain fragments (scFv) from a diverse library generated from mice immunized with a variant peptide of the transcription factor GCN4 dimerization domain. After three rounds of ribosome display, positive scFvs were isolated and characterized. Several different scFvs were selected, but those in the largest group were closely related to each other and differed in 0 to 5 amino acid residues with respect to their consensus sequence, the likely common progenitor. The best scFv had a dissociation constant of (4 +/- 1) x 10(-11) M, measured in solution. One amino acid residue in complementarity determining region L1 was found to be responsible for a 65-fold higher affinity than the likely progenitor. It appears that this high-affinity scFv was selected from the mutations occurring during ribosome display in vitro, and that this constitutes an affinity maturation inherent in this method. The in vitro-selected scFvs could be functionally expressed in the Escherichia coli periplasm with good yields or prepared by in vitro refolding. Thus, ribosome display can be a powerful methodology for in vitro library screening and simultaneous sequence evolution.
authors.affiliation: Biochemisches Institut, Universität Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.
authors.forename: J
authors.initials: J
authors.lastname: Hanes
authors.forename: L
authors.initials: L
authors.lastname: Jermutus
authors.forename: S
authors.initials: S
authors.lastname: Weber-Bornhauser
authors.forename: H R
authors.initials: HR
authors.lastname: Bosshard
authors.forename: A
authors.initials: A
authors.lastname: Plückthun
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000915:Antibody Affinity; D002846:Chromatography, Affinity; D004797:Enzyme-Linked Immunosorbent Assay; D007128:Immunoglobulin Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007147:Immunoglobulin Light Chains / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D019151:Peptide Library; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012996:Solutions; D013154:Spleen / Q000276:immunology; D014158:Transcription, Genetic
chemical_list: D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D012333:RNA, Messenger; D011994:Recombinant Proteins; D012996:Solutions