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光合成細菌を宿主とする膜タンパク質の高効率大量発現系の構築

以光合细菌为宿主构建高效大规模膜蛋白表达系统

基本信息

批准号：
20657023
负责人：
佐伯和彦
金额：
$ 2.05万
依托单位：
Nara Women's University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Challenging Exploratory Research
财政年份：
2008
资助国家：
日本
起止时间：
2008 至 2010
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-20657023/
关键词：
光合成細菌微好気条件タンパク質精製

项目摘要

本研究では、光合成細菌が暗所・微好気条件下でも多量の細胞内膜陥入構造(クロマトフォア)を形成し大量の光化学系関連の膜タンパク質を蓄積しうる点に着目して、膜含量自体を増大させた上で光化学系の替わりに外来の膜タンパク質を安定かつ多量に発現させることを目指した。Sec分泌系を用いて発現させた場合に引き起こされる基質輸送体や電子伝達系、ATP合成酵素などの機能発現との競合を避けるために、膜配置・分泌シグナルとして、枯草菌由来のMISTIC(Membrane Integrating Sequence for Translation of Integral Membrane Protein Constructs)タンパク質を用いることとし、MISTIC人工遺伝子を光合成細菌Rhodobacter capsulatusのコドン頻度に合わせて作製した。クロマトフォアを形成しない好気条件下で目的の膜タンパク質を発現せず、微好気条件でのみ多量に発現させるために、酸素分圧の低下により制御を受けるpufプロモーターを人工遺伝子の上流に配置した。また、下流にはρ因子非依存性の転写終結を惹起する人工配列を配置した。なお、産物のアミノ末端側にヘキサHis配列とカルボキシ末端側に血液凝固系のタンパク質分解酵素トロンビン認識配列などを付加して、発現させる膜タンパク質の精製を容易にした。人工遺伝子とのin-frame融合により、GFPならびにR.capsulatus自身のRnfの過剰発現を確認した。安定発現のため宿主にpuhBとdegPの変異を導入したが、効果は限定的であった。また、長時間の培養時に宿主株の示す溶菌傾向に変化は認められなかった。一方、膜画分から標的タンパク質を可溶化・精製の際に、カロチノイド色素が標的と類似の挙動を示した。今後、溶菌傾向と色素生成能を軽減させた株を作出する必要が有る。

In this study, a large number of photochemical systems were formed under the condition that the photosynthetic bacteria were in the dark. In this study, a large number of photochemical systems were formed under the condition that the photosynthetic bacteria were in the dark. In this study, a large number of photochemical systems were formed under the conditions of large amounts of photochemical bacteria and photosynthetic bacteria. The Sec secretory system was used to detect the enzyme complex, and the ATP synthetic enzyme enzyme assay was used to detect the immune response, the membrane configuration was used to secrete the enzyme, and the Bacillus subtilis was isolated from Mistic (Membrane Integrating Sequence for Translation of Integral Membrane Protein Constructs). The photosynthetic bacteria of MISTIC artificial mackerel were used as photosynthetic bacteria (Rhodobacter capsulatus) in combination with each other. Under good conditions, the purpose of the membrane is good, the quantity is large, the content of acid is low, and the content of acid is low. It is controlled by the puf device, the artificial device, the upstream configuration. The non-dependence of the ρ factor is caused by the manual arrangement of the ρ factor. In the end of the His, the blood clotting system, the decomposing enzyme, the biodegradable enzyme, the biodegradable enzyme, the membrane, the blood clotting system, the blood clotting system, the decomposing enzyme, the biodegradable enzyme and the membrane. Manual in-frame fusion test, GFP fusion test, R.capsulatus self-validation, Rnf verification can be realized. Diazepam is available to the host, puhB, degP, hosts, hosts and hosts. When the host strain is incubated for a long period of time, the host strain is shown to be bacteriolytic. On the other hand, the type of the color pigment of the film painting is similar to that of the film painting. In the future, the bacteriolytic agent will make a necessary contribution to the production of pigment.