Development of assays and antibodies for functional analysis of taste receptors.

• Project/Area Number: 26750365
• Research Category: Grant-in-Aid for Young Scientists (B)
• Allocation Type: Multi-year Fund
• Research Field: Biomolecular chemistry
• Research Institution: Ehime University
• Principal Investigator: Hiroyuki Takeda 愛媛大学, プロテオサイエンスセンター, 准教授 (40609393)
• Project Period (FY): 2014-04-01 – 2016-03-31
• Project Status: Completed (Fiscal Year 2015)
• Budget Amount: ¥3,770,000 (Direct Cost: ¥2,900,000、Indirect Cost: ¥870,000); Fiscal Year 2015: ¥2,210,000 (Direct Cost: ¥1,700,000、Indirect Cost: ¥510,000); Fiscal Year 2014: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000)
• Keywords: モノクローナル抗体 / GPCR / 抗原 / 無細胞タンパク質合成 / 味覚受容体 / リポソーム / 膜タンパク質 / 相互作用 / コムギ無細胞系 / 脂質 / コムギ無細胞合成 / プロテオリポソーム / 抗体

Outline of Final Research Achievements

T1R family GPCR is sweet/umami receptor. Overexpression system and specific antibodies of T1R family were not established. In this study, we synthesized T1R1 and T1R3 using cell-free membrane protein expression system. Using cell-free synthesized T1Rs, we developed specific monoclonal antibodies. In addition to anti-T1R antibody, we also obtained monoclonal antibody against asolectin lipid. Anti-asolectin antibody bound to asolectin liposome, however it did not EggPC liposome. We developed an assay method to detect interaction between liposomes. Monoclonal antibodies and assay methods developed in this study may contribute to functional analysis of membrane proteins including taste receptors.

Research Products

• [Journal Article] 無細胞技術を基盤とした抗GPCR モノクローナル抗体作製技術の開発 (2016)
  • Author(s): 竹田浩之、澤崎達也
  • Journal Title: 月刊バイオインダストリー Volume: 32 Pages: 56-62
• [Journal Article] Production of monoclonal antibodies against GPCR using cell-free synthesized GPCR antigen and biotinylated liposome-based interaction assay (2015)
  • Author(s): Takeda H, Ogasawara T, Ozawa T, Muraguchi A, Jih PJ, Morishita R, Uchigashima M, Watanabe M, Fujimoto T, Iwasaki T, Endo Y, Sawasaki T
  • Journal Title: Sci Rep Volume: 5 Issue: 1 Pages: 11333-11333
  • DOI: 10.1038/srep11333
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Na,K-ATPaseα3 is a death target of alzheimer patient amyloid-β assembly. (2015)
  • Author(s): Ohnishi T, ..., Matsui K, ..., Hoshi M （計35名, 記載欄不足）
  • Journal Title: Proc Natl Acad Sci U S A Volume: 112 Issue: 32 Pages: 4465-4474
  • DOI: 10.1073/pnas.1421182112
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Claudin-1 Binder Enhances Epidermal Permeability in a Human Keratinocyte Model (2015)
  • Author(s): Nakajima M, Nagase S, Iida M, Takeda S, Yamashita M, Watari A, Shirasago Y, Fukasawa M, Takeda H, Sawasaki T, Yagi K, Kondoh M
  • Journal Title: J Pharmacol Exp Ther Volume: 354 Issue: 3 Pages: 440-447
  • DOI: 10.1124/jpet.115.225391
  • Peer Reviewed
• [Journal Article] リポソーム添加型コムギ無細胞合成系による膜タンパク質合成とその利用 (2015)
  • Author(s): 竹田浩之、野澤彰、澤崎達也
  • Journal Title: ファルマシア Volume: 51 Pages: 770-774
  • NAID: 130007448253
• [Presentation] Production of monoclonal antibodies against GPCR using cell-free synthesized GPCR antigen and biotinylated liposome-based interaction assay. (2015)
  • Author(s): Takeda H, Ogasawara T, Ozawa T, Muraguchi A, Jih PJ, Morishita R, Uchigashima M, Watanabe M, Fujimoto T, Iwasaki T, Endo Y, Sawasaki T.
  • Organizer: GPCR work shop
  • Place of Presentation: Kona, Big island Hawaii, Hi, USA
  • Year and Date: 2015-12-01
  • Int'l Joint Research
• [Presentation] Production of monoclonal antibodies against GPCR using cell-free synthesized GPCR antigen and biotinylated liposome-based interaction assay (2015)
  • Author(s): 2) Takeda H, Ogasawara T, Ozawa T, Muraguchi A, Jih PJ, Morishita R, Uchigashima M, Watanabe M, Fujimoto T, Iwasaki T, Endo Y, Sawasaki T
  • Organizer: Protein Island Matsuyama international symposium
  • Place of Presentation: 松山市男女共同参画推進センター（愛媛県・松山市）
  • Year and Date: 2015-09-25
  • Int'l Joint Research
• [Presentation] 栄谷 紘一、竹田 浩之、小澤 龍彦、村口 篤、三坂 巧、森下 了、澤崎 達也 (2015)
  • Author(s): コムギ無細胞合成系を基盤とした旨味/甘味受容体タンパク質の合成とモノクローナル抗体の作製
  • Organizer: 日本農芸化学会
  • Place of Presentation: 岡山大学
  • Year and Date: 2015-03-26 – 2015-03-29
• [Presentation] Cell-free Synthesized G-protein-coupled Receptor Proteoliposome is Efficient for Biochemical Analysis and Monoclonal Antibody Development (2014)
  • Author(s): Hiroyuki Takeda, Tomio Ogasawara, Tatsuhiko Ozawa, Atsushi Muraguchi, Yaeta Endo, Tatsuya Sawasaki
  • Organizer: Discovery on Target 2014
  • Place of Presentation: 米国ボストン
  • Year and Date: 2014-10-08 – 2014-10-10
• [Presentation] コムギ無細胞タンパク質合成系を基盤とした 甘味受容体機能構造解析のための技術開発 (2014)
  • Author(s): 栄谷紘一・竹田浩之・小澤龍彦・村口篤・澤崎達也
  • Organizer: GPCR研究会
  • Place of Presentation: 日本科学未来館
  • Year and Date: 2014-05-09 – 2014-05-10
• [Remarks] プロテオサイエンスセンター トピックス
  • URL: http://www.pros.ehime-u.ac.jp/topics/detail.php?serial=76
• [Remarks] 愛媛大学プレスリリース
  • URL: http://www.pros.ehime-u.ac.jp/upfile/1434934243_01.pdf