Factors involved in difficult-to-express protein production and their characterization

2023 Fiscal Year Final Research Report

• Project/Area Number: 21K04795
• 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: University of Shizuoka
• Principal Investigator: Kawarasaki Yasuaki 静岡県立大学, 食品栄養科学部, 准教授 (80303585)
• Co-Investigator(Kenkyū-buntansha): 田中 瑞己 東京農工大学, (連合)農学研究科(研究院), 准教授 (70803344)
• Project Period (FY): 2021-04-01 – 2024-03-31
• Keywords: 組換え蛋白質 / 遺伝子機能評価 / 酵母転写因子 / 出芽酵母発現系 / 難生産性蛋白質 / プロモーター / 遺伝子発現制御

Outline of Final Research Achievements

A novel yeast protein expression system for "difficult-to-express" proteins is established in this study. The system features a combination of a yeast host and a promoter sequence on the expression vector-- i.e., ygr067C-disrupted yeast and its promoter.
The biological characteristics of the gene and its protein have also delineated in this study. The Ygr067C encodes uncharacterized transcription repressor. The expression of the gene is 1) late diauxic phase specific, 2) auto-regulative, and 3) transient in anaerobic condition. The translated protein of the gene has been unstable in the cell, and degraded in a single round of cell-cycle. The genes of which expression are regulated by the Ygr067C have also been analyzed. The promoter of the gene has also been characterized and the activation condition is optimized.

Free Research Field

生物工学

Academic Significance and Societal Importance of the Research Achievements

活性型での発現が困難であるため解析が困難である蛋白質は多い。このような難生産性蛋白質に対し、今回、取り扱いが容易な出芽酵母を用いて効率的な発現系を構築することができた。遺伝子発現に際し、この系では誘導物質の濃度、添加時期などの最適化は不要であるため、難生産性が予想される蛋白質の発現系としてファーストチョイスになり得る、極めて簡便な発現系が構築できたと言える。特に多数のパラログがあり個別の遺伝子およびその遺伝子産物の評価が困難な、例えば菌類の酵素遺伝子群の機能解析を非常に容易にすることができた。