Construction of highly efficient completely cell-free protein synthesis system using insect extract

2021 Fiscal Year Final Research Report

• Project/Area Number: 19K05176
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Review Section: Basic Section 27040:Biofunction and bioprocess engineering-related
• Research Institution: Okinawa National College of Technology
• Principal Investigator: Ito Masaaki 沖縄工業高等専門学校, 生物資源工学科, 教授 (00395659)
• Project Period (FY): 2019-04-01 – 2022-03-31
• Keywords: 無細胞タンパク質合成系 / カイコ / 昆虫培養細胞 / 翻訳促進配列

Outline of Final Research Achievements

In this study, we first investigated the enhancer sequence, succeeded in improving the efficiency of experimental operations in the silkworm cell-free protein synthesis system, and were able to construct a completely cell-free protein synthesis system. Next, in order to improve the amount of synthesis, the conditions for preparing the extract derived from the silk moth posterior silk gland were examined. As a result, we established a method for preparing an extract having a synthetic ability of 100 μg / ml or more when the synthesis of β-galactosidase was used as a control. In addition, the optimum chelating agent concentration of the link method, which saves the trouble of RNA purification using the extract, was examined. As a result, it was clarified that the amount of synthesis was the highest in EDTA 0.5 mM. As a result, we succeeded in improving the amount of synthesis in the silk moth cell-free protein synthesis system.

Free Research Field

タンパク質工学

Academic Significance and Societal Importance of the Research Achievements

本研究は、試薬キットとして実用化されたが未だマイナーなタンパク質生産技術である昆虫無細胞系の合成効率および利便性の向上を図ることで「創薬研究・ポストゲノム研究に使われる技術」にすることを目的とした。
これまで申請者が開発してきた昆虫無細胞系は、カイコ幼虫や昆虫培養細胞が高いタンパク質合成能を有することに着目して研究をすすめ、昆虫を利用した科学技術革新を目指しており学術的に意義がある。また、この系は、動物由来の無細胞系の中で高い合成能を有しており、各種翻訳後修飾も哺乳動物と同様であることから極めて有用であり、利便性を高め、さらに合成量を高めたことの意義は大きい。