Construction of E. coli expression system for eukaryotic membrane proteins

• Project/Area Number: 15K14475
• Research Category: Grant-in-Aid for Challenging Exploratory Research
• Allocation Type: Multi-year Fund
• Research Field: Functional biochemistry
• Research Institution: Kyoto University
• Principal Investigator: Imai Tomoya 京都大学, 生存圏研究所, 准教授 (90509142)
• Co-Investigator(Kenkyū-buntansha): 石水 毅 立命館大学, 生命科学部, 准教授 (30314355) ; 中島 啓介 沖縄科学技術大学院大学, マリンゲノミックスユニット, 研究員 (10422924)
• Co-Investigator(Renkei-kenkyūsha): SUZUKI Shiro 京都大学, 生存圏研究所, 助教 (70437268)
• Project Period (FY): 2015-04-01 – 2017-03-31
• Project Status: Completed (Fiscal Year 2016)
• Budget Amount: ¥3,900,000 (Direct Cost: ¥3,000,000、Indirect Cost: ¥900,000); Fiscal Year 2016: ¥2,080,000 (Direct Cost: ¥1,600,000、Indirect Cost: ¥480,000); Fiscal Year 2015: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
• Keywords: 膜タンパク質 / 真核生物 / 細胞膜脂質 / ホスファチジルコリン / 大腸菌発現系 / 膜タンパク質発現 / 多糖合成酵素

Outline of Final Research Achievements

The cell membrane of eukaryotic cells includes phosphatidyl choline (PC) whereas that in E. coli cells does not have PC but phosphatidyl ethanolamine (PE). Given this lipid composition, we tested the idea to use Escherichia coli expression system for expressing eukaryotic membrane protein by using a transformed E. coli that produces PC in its cell membrane.
We successfully produced an E .coli transformant producing PC in its cell membrane. Thsi transformant was used for expressing cellulose synthase complex of Gluconacetobacter (a bacterium producing PC), CesA - the catalytic subunit of cellulose synthase - of a green algae, CesA of a tunicate, and a glycosyltransferase from plant. Although this strategy did not work well in most of the experiments, one of the experiments for expressing green algae CesA showed the expression in the cell membrane of E. coli transformant. Further study will be necessary for reproducible result for concluding if this strategy is realistic.