title: Selection, characterization and x-ray structure of anti-ampicillin single-chain Fv fragments from phage-displayed murine antibody libraries.
abstract: Single-chain Fv (scFv) antibody libraries were constructed from mice immunized with an ampicillin-bovine serum albumin conjugate. Several antibodies with specificity for intact ampicillin were selected by phage display and characterized. The antibody scFv fragment aL2 binds to intact ampicillin and shows no detectable cross-reactivity with hydrolyzed ampicillin. We determined the X-ray structures of two crystal forms of w.t. aL2, which differ mainly in the side-chain conformation of Trp H109 (according to a new consensus nomenclature Kabat residue number H95) in the extremely short (three residues) CDR H3 and the presence or absence of a well-resolved molecule of 2-methyl-pentane-2,4-diol in the bottom of the binding pocket. Attempts to co-crystallize aL2 with its antigen or to diffuse ampicillin into the wild-type aL2 crystals were unsuccessful, since crystal contacts obstruct the binding pocket. However, a mutant with two point mutations near the N terminus (Gln H6 replaced by Glu and Ala H10 (Kabat H9) replaced by Gly) crystallized in a form compatible with antigen-binding. Although the mutations affect the conformation of framework I, the conformations of the binding pocket of the uncomplexed wild-type aL2 and of the mutant complex were almost identical. The structure explains the specificity of the antibody for intact ampicillin and the degree of cross-reactivity of aL2 with a wide variety of ampicillin analogs. This antibody system will be very useful as a diagnostic reagent for antibiotics use and abuse, as a model for the effect of expression of antibiotic binding molecules in Escherichia coli, and for directed evolution towards high antibiotic resistance.
authors.affiliation: Biochemisches Institut der Universität Zürich, Winterthurerstrasse 190, Zürich, CH-8057, Switzerland.
authors.forename: J
authors.initials: J
authors.lastname: Burmester
authors.forename: S
authors.initials: S
authors.lastname: Spinelli
authors.forename: L
authors.initials: L
authors.lastname: Pugliese
authors.forename: A
authors.initials: A
authors.lastname: Krebber
authors.forename: A
authors.initials: A
authors.lastname: Honegger
authors.forename: S
authors.initials: S
authors.lastname: Jung
authors.forename: B
authors.initials: B
authors.lastname: Schimmele
authors.forename: C
authors.initials: C
authors.lastname: Cambillau
authors.forename: A
authors.initials: A
authors.lastname: Plückthun
mesh_terms: D000595:Amino Acid Sequence; D000667:Ampicillin / Q000276:immunology* / Q000378:metabolism; D000818:Animals; D000915:Antibody Affinity; D000918:Antibody Specificity; D001666:Binding Sites, Antibody; D022801:Complementarity Determining Regions / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D003460:Crystallization; D018360:Crystallography, X-Ray; D018604:Epitope Mapping; D006241:Haptens / Q000276:immunology; D006860:Hydrogen Bonding; D007114:Immunization; D007128:Immunoglobulin Fragments / Q000737:chemistry* / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D019151:Peptide Library; D011487:Protein Conformation; D015202:Protein Engineering; D016415:Sequence Alignment; D012710:Serum Albumin, Bovine
chemical_list: D022801:Complementarity Determining Regions; D006241:Haptens; D007128:Immunoglobulin Fragments; D019151:Peptide Library; D012710:Serum Albumin, Bovine; D000667:Ampicillin