Proteoliposome-based selection of a recombinant antibody fragment against the human M2 muscarinic acetylcholine receptor

Suharni, Yayoi Nomura, Takatoshi Arakawa, Tomoya Hino, Hitomi Abe, Yoshiko Nakada-Nakura, Yumi Sato, Hiroko Iwanari, Mitsunori Shiroishi, Hidetsugu Asada, Tatsuro Shimamura, Takeshi Murata, Takuya Kobayashi, Takao Hamakubo, So Iwata, Norimichi Nomura

Research output: Contribution to journalArticlepeer-review

8 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)378-385
Number of pages8
JournalMonoclonal Antibodies in Immunodiagnosis and Immunotherapy
Volume33
Issue number6
DOIs
Publication statusPublished - Dec 1 2014

All Science Journal Classification (ASJC) codes

  • Immunology and Allergy
  • Immunology

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