Crystallization of stabilized leukotriene B4 receptor mutant and monoclonal antibody complex.

• Project/Area Number: 23770133
• Research Category: Grant-in-Aid for Young Scientists (B)
• Allocation Type: Multi-year Fund
• Research Field: Structural biochemistry
• Research Institution: The Institute of Physical and Chemical Research
• Principal Investigator: HORI Tetsuya 独立行政法人理化学研究所, 宮野構造生物物理研究室, 専任研究員 (20344054)
• Project Period (FY): 2011 – 2012
• Project Status: Completed (Fiscal Year 2012)
• Budget Amount: ¥4,550,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥1,050,000); Fiscal Year 2012: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000); Fiscal Year 2011: ¥2,990,000 (Direct Cost: ¥2,300,000、Indirect Cost: ¥690,000)
• Keywords: ロイコトリエンB_4 受容体 / G-タンパク質共役型受容体 / メタノール資化酵母 / 安定化変異体 / モノクローナル抗体 / 大量発現 / 精製 / 結晶化 / ロイコトリエンB4受容体 / 安定化 / モノクロ抗体作成 / GPCR / 熱安定化 / 大量調製

Research Abstract

For the crystallization of leukotriene B_4 receptor (BLT1), the stabilized mutant was constructed and the anti-BLT1 monoclonal antibody which recognized three-dimensional structure of BLT1 was generated. The rationally designed two point mutations were improved the thermal-stability by 5 ℃ and the expression level of active form BLT1 by 6 times (Bmax=311 pmol/mg). The antibody which bound the none-denatured BLT1 but did not recognize the SDS-denatured BLT1 was generated. The crystallization of BLT1-Fab complex has been promoted.

Research Products

• [Presentation] Thermostabilization and generation of monoclonal antibody to leukotriene B_4 receptor (BLT1) for crystallization, (2011)
  • Author(s): Hori T., Sugahara M., Nakamura M., Yokomizo T., Shimizu T., and Miyano M.
  • Organizer: GPCR Workshop 2011
  • Place of Presentation: マウイ
  • Year and Date: 2011-12-05
• [Presentation] 結晶化のためのロイコトリエンB_4受容体(BLT1)の熱安定化と抗BLT1モノクローナル抗体の作製 (2011)
  • Author(s): 堀 哲哉、菅原光明、中村元直、横溝岳彦、清水孝雄、宮野雅司
  • Organizer: 第84回日本生化学会大会(BMB2011)
  • Place of Presentation: 京都
  • Year and Date: 2011-09-22
• [Presentation] Thermostabilization and generation of monoclonal antibody to leukotriene B4 receptor (BLT1) for crystallization (2011)
  • Author(s): 堀 哲哉
  • Organizer: GPCR workshop 2011
  • Place of Presentation: マウイ、ハワイ
• [Presentation] 結晶化のためのロイコトリエンB4受容体(BLT1)の熱安定化と抗BLT1モノクローナル抗体の作製 (2011)
  • Author(s): 堀 哲哉
  • Organizer: 日本生化学会年会
  • Place of Presentation: 京都国際会館