title: Proteoliposome-based selection of a recombinant antibody fragment against the human M2 muscarinic acetylcholine receptor.
abstract: The development of antibodies against human G-protein-coupled receptors (GPCRs) has achieved limited success, which has mainly been attributed to their low stability in a detergent-solubilized state. We herein describe a method that can generally be applied to the selection of phage display libraries with human GPCRs reconstituted in liposomes. A key feature of this approach is the production of biotinylated proteoliposomes that can be immobilized on the surface of streptavidin-coupled microplates or paramagnetic beads and used as a binding target for antibodies. As an example, we isolated a single chain Fv fragment from an immune phage library that specifically binds to the human M2 muscarinic acetylcholine receptor with nanomolar affinity. The selected antibody fragment recognized the GPCR in both detergent-solubilized and membrane-embedded forms, which suggests that it may be a potentially valuable tool for structural and functional studies of the GPCR. The use of proteoliposomes as immunogens and screening bait will facilitate the application of phage display to this difficult class of membrane proteins.
authors.affiliation: 1 Department of Cell Biology, Graduate School of Medicine, Kyoto University , Sakyo-ku, Kyoto, Japan .
authors.lastname: Suharni
authors.forename: Yayoi
authors.initials: Y
authors.lastname: Nomura
authors.forename: Takatoshi
authors.initials: T
authors.lastname: Arakawa
authors.forename: Tomoya
authors.initials: T
authors.lastname: Hino
authors.forename: Hitomi
authors.initials: H
authors.lastname: Abe
authors.forename: Yoshiko
authors.initials: Y
authors.lastname: Nakada-Nakura
authors.forename: Yumi
authors.initials: Y
authors.lastname: Sato
authors.forename: Hiroko
authors.initials: H
authors.lastname: Iwanari
authors.forename: Mitsunori
authors.initials: M
authors.lastname: Shiroishi
authors.forename: Hidetsugu
authors.initials: H
authors.lastname: Asada
authors.forename: Tatsuro
authors.initials: T
authors.lastname: Shimamura
authors.forename: Takeshi
authors.initials: T
authors.lastname: Murata
authors.forename: Takuya
authors.initials: T
authors.lastname: Kobayashi
authors.forename: Takao
authors.initials: T
authors.lastname: Hamakubo
authors.forename: So
authors.initials: S
authors.lastname: Iwata
authors.forename: Norimichi
authors.initials: N
authors.lastname: Nomura
mesh_terms: D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D006801:Humans; D007128:Immunoglobulin Fragments / Q000096:biosynthesis* / Q000276:immunology*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D019151:Peptide Library; D011485:Protein Binding; D011510:Proteolipids / Q000096:biosynthesis* / Q000378:metabolism*; D043585:Receptor, Muscarinic M2 / Q000235:genetics / Q000276:immunology*; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology*; D012441:Saccharomyces cerevisiae
chemical_list: D007128:Immunoglobulin Fragments; D019151:Peptide Library; D011510:Proteolipids; D043585:Receptor, Muscarinic M2; D011994:Recombinant Proteins; C020485:proteoliposomes