Technologicaldevelopment for construction of synthetic proteins that facilitate membrane protein crystallization

2012 Fiscal Year Final Research Report

• Project/Area Number: 22570114
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Structural biochemistry
• Research Institution: Kyoto University
• Principal Investigator: NOMURA Norimichi 京都大学, 大学院・医学研究科, 助教 (10314246)
• Co-Investigator(Renkei-kenkyūsha): IWATA So 京都大学, 大学院・医学研究科, 教授 (60452330)
• Project Period (FY): 2010 – 2012
• Keywords: 膜蛋白質 / X線結晶構造解析 / 結晶化リガンド / 抗体フラグメント / ファージディスプレイ

Research Abstract

Co-crystallization of membrane proteins with antibody fragments may emerge as a general tool to facilitate crystal growth and improve crystal quality. The bound antibody fragment enlarges the hydrophilic part of the mostly hydrophobic membrane protein, thereby increasing the interaction area for possible protein-protein contacts in the crystal. To serve as a ‘crystallizing ligand’, the antibody fragment has to recognize an epitope that is only present in the native conformation (and not in the denatured state) of the membrane protein, bind with high affinity, and form stable and rigid complexes. In this project, we developed a fast and reliable method for generating crystallizing ligands against various mammalian G-protein coupled receptors (GPCRs) and transporters in a phage display format. Using this method, the crystal structure of a mammalian facilitative glucose transporter complexed with a Fv fragment has been determined at 3.5Åresolution.

Research Products

• [Journal Article] G-protein-coupled receptor inactivation by an allosteric inverse-agonist antibody (2012)
  • Author(s): Hino T, Arakawa T, Iwanari H, Yurugi-Kobayashi T, Ikeda-Suno C, Nakada-Nakura Y, Kusano-Arai O, Weyand S, Shimamura T, Nomura N, Cameron AD, Kobayashi T, Hamakubo T, Iwata S, Murata T
  • Journal Title: Nature Volume: 482(7384) Pages: 237-40
  • DOI: DOI:10.1038/nature10750
  • Peer Reviewed
• [Journal Article] Platform for the rapid construction and evaluation of GPCRs for crystallography in Saccharomyces cerevisiae (2012)
  • Author(s): Shiroishi M, Tsujimoto H, Makyio H, Asada H, Yurugi-Kobayashi T, Shimamura T, Murata T, Nomura N, Haga T, Iwata S, Kobayashi T
  • Journal Title: Microb Cell Fact Volume: 11 Pages: 78
  • DOI: DOI:10.1186/1475-2859-11-78
  • Peer Reviewed
• [Journal Article] Cloning, expression and purification of the anion exchanger 1 homologue from the basidiomycete Phanerochaete chrysosporium (2011)
  • Author(s): Tokuda N, Igarashi K, Shimamura T, Yurugi-Kobayashi T, Shiroishi M, Ito K, Sugawara T, Asada H, Murata T, Nomura N, Iwata S, Kobayashi T
  • Journal Title: Protein Expr Purif Volume: 79(1) Pages: 81-7
  • DOI: DOI:10.1016/j.pep.2011.04.006
  • Peer Reviewed
• [Journal Article] Evaluation of the Pichia pastorisexpression system for the production of GPCRs for structural analysis. (2011)
  • Author(s): Asada H, Uemura T, Yurugi-Kobayashi T, Shiroishi M, Shimamura T, Tsujimoto H, Ito K, Sugawara T, Nakane T, Nomura N, Murata T, Haga T, Iwata S, Kobayashi T
  • Journal Title: Microb Cell Fact Volume: 22 Pages: 24
  • DOI: DOI:10.1186/1475-2859-10-24
  • Peer Reviewed
• [Presentation] 膜蛋白質のX線結晶構造解析を支援する抗体工学技術の確立 (2011)
  • Author(s): 野村紀通,他
  • Organizer: 第84回日本生化学会大会 シンポジウム「多様な生物種における膜タンパク質の機能理解を目指した新戦略」
  • Place of Presentation: 京都
  • Year and Date: 2011-09-24
• [Patent(Industrial Property Rights)] 抗ヒトバンド3モノクローナル抗体 (2011)
  • Inventor(s): 小林拓也 他
  • Industrial Property Rights Holder: 科学技術振興機構
  • Industrial Property Number: 特願2011-246364
  • Filing Date: 2011-11-10