Construction of a cell-free reconstitution system for GPCRs using a novel membrane insertion promoter

2023 Fiscal Year Final Research Report

• Project/Area Number: 22K19099
• Research Category: Grant-in-Aid for Challenging Research (Exploratory)
• Allocation Type: Multi-year Fund
• Review Section: Medium-sized Section 37:Biomolecular chemistry and related fields
• Research Institution: Saitama University
• Principal Investigator: Tozawa Yuzuru 埼玉大学, 理工学研究科, 教授 (90363267)
• Project Period (FY): 2022-06-30 – 2024-03-31
• Keywords: 無細胞翻訳系 / GPCR / ペプチドアプタマー / 進化工学 / プロスタグランジン受容体

Outline of Final Research Achievements

In this project, we constructed a reconstitution system for the G protein coupling receptor (GPCR), an important target membrane protein in drug discovery, and an evolutionary engineering system to select peptide aptamers that specifically bind to this GPCR efficiently. Analysis of Asterix, which is assumed to hold the key to the efficiency of GPCR reconstitution, revealed its autonomous membrane insertion and dimer formation abilities. However, we could not confirm the effect of promoting membrane insertion by creating a chimeric molecule with GPCR. On the other hand, we reconstituted GPCR purified from cells into nanodiscs, and by using this as bait for evolutionary engineering, we succeeded in obtaining eight artificial antibodies from an antibody library that can recognize the extracellular domain of GPCR. We even confirmed that these antibodies actually bind to the cell surface of pancreatic cancer cells and other cancer cells.

Free Research Field

無細胞翻訳系

Academic Significance and Societal Importance of the Research Achievements

本研究成果は，GPCRの中でも膵臓がん細胞などのマーカー候補プロスタグランジン受容体EP4に対し，細胞外領域を特異的に認識可能な人工抗体を取得できた点で，学術的・社会的意義が大きい．良質な標的分子を準備さえすれば，無細胞翻訳系にもとづく進化分子工学の手法により，創薬展開に適する人工抗体などを効率的に選抜可能になった．産業応用上のゴールは，需要が大きな創薬用人工抗体の効率的取得システムの提供であり，社会貢献に直結する成果と考えている．また，学術的にもGPCRを含む膜タンパク質の品質管理の鍵を握るAsterixの基本的な分子挙動について生化学的な知見が得られた点では進捗があったと考えている．