title: Rapid identification of recombinant Fabs that bind to membrane proteins.
abstract: Crystallographic studies of membrane proteins have been steadily increasing despite their unique physical properties that hinder crystal formation. Co-crystallization with antibody fragments has emerged as a promising solution to obtain diffraction quality crystals. Antibody binding to the target membrane protein can yield a homogenous population of the protein. Interantibody interactions can also provide additional crystal contacts, which are minimized in membrane proteins due to micelle formation around the transmembrane segments. Rapid identification of antibody fragments that can recognize native protein structure makes phage display a valuable method for crystallographic studies of membrane proteins. Methods that speed the reliable characterization of phage display selected antibody fragments are needed to make the technology more generally applicable. In this report, a phage display biopanning procedure is described to identify Fragments antigen binding (Fabs) for membrane proteins. It is also demonstrated that Fabs can be rapidly grouped based on relative affinities using enzyme linked immunosorbent assay (ELISA) and unpurified Fabs. This procedure greatly speeds the prioritization of candidate binders to membrane proteins and will aid in subsequent structure determinations.
authors.affiliation: Department of Pharmaceutical Chemistry, University of California, San Francisco, 600, 16th Street, San Francisco, CA 94158, USA. jungmin.kim@ucsf.edu
authors.forename: Jungmin
authors.initials: J
authors.lastname: Kim
authors.forename: Robert M
authors.initials: RM
authors.lastname: Stroud
authors.forename: Charles S
authors.initials: CS
authors.lastname: Craik
mesh_terms: D001666:Binding Sites, Antibody; D019921:Biotinylation; D002846:Chromatography, Affinity; D003460:Crystallization; D003461:Crystallography / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D055767:Immobilized Proteins / Q000737:chemistry / Q000302:isolation & purification; D007128:Immunoglobulin Fragments / Q000737:chemistry* / Q000302:isolation & purification; D008565:Membrane Proteins / Q000737:chemistry* / Q000302:isolation & purification; D019151:Peptide Library; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry* / Q000302:isolation & purification
chemical_list: D055767:Immobilized Proteins; D007128:Immunoglobulin Fragments; D008565:Membrane Proteins; D019151:Peptide Library; D011994:Recombinant Proteins