Complex protein fishing method using proximity dependent reaction of modified transglutaminase

2018 Fiscal Year Final Research Report

• Project/Area Number: 16K06865
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Biofunction/Bioprocess
• Research Institution: Akita University
• Principal Investigator: Gotoh Takeshi 秋田大学, 理工学研究科, 教授 (10215494)
• Research Collaborator: Yokota Saki
• Project Period (FY): 2016-04-01 – 2019-03-31
• Keywords: トランスグルタミナーゼ / プロテオミクス / 組換えタンパク質生産

Outline of Final Research Achievements

Two approaches were examined to prepare a fusion protein of transglutaminase (TGase) and bait protein, to determine the possibility that the TGase-bait fusion protein may be used to explore unknown proteins (prey) that bind to the known protein (bait), by proximity-dependent labeling reaction of TGase. First, a modified TGase, in which a TGase-reactive peptide was attached to the C-terminus through a His tag, was prepared and used to couple with a bait protein by its own catalytic activity. However, this approach did not work well probably due to very low concentrations of the proteins. Then, a recombinant E. coli, which was engineered to secrete separately a TGase-FRB fusion and its pro peptide to the periplasm, was constructed. As a result, the TGase fusion protein with FRB attached to the C-terminus could be produced directly in E. coli periplasm and showed an enough TGase activity.

Free Research Field

生物化学工学

Academic Significance and Societal Importance of the Research Achievements

細胞や組織に発現しているタンパク質の動態を把握し，それらのタンパク質が織りなす相互作用の実態を解析してこれを系統的・包括的に捉えようとするプロテオミクス研究の重要性が近年高かまっている。本研究では，TGaseを利用した近接依存標識反応によって未知タンパク質を標識・同定するために必要な，未知タンパク質と相互作用する既知タンパク質とTGaseの融合体を調製する方法について，有益な方策を提案した。