- 著者
- Yuki Sudo Rikou Tanaka Toshitatsu Kobayashi Naoki Kamo Toshiyuki Kohno Chojiro Kojima
- 出版者
- The Biophysical Society of Japan
- 雑誌
- BIOPHYSICS (ISSN:13492942)
- 巻号頁・発行日
- vol.7, pp.51-58, 2011 (Released:2011-06-18)
- 参考文献数
- 34
An approach of cell-free synthesis is presented for the functional expression of transmembrane proteins without the need of refolding. The transmembrane region of the pharaonis halobacterial transducer protein, pHtrII, was translated with various large soluble tags added (thioredoxin, glutathione S-transferase, green fluorescent protein and maltose binding protein). In this system, all fusion pHtrII were translated in a soluble fraction, presumably, forming giant micelle-like structures. The detergent n-dodecyl-β-D-maltoside was added for enhancing the solubilization of the hydrophobic region of pHtrII. The activity of the expressed pHtrII, having various tags, was checked using a pull-down assay, using the fact that pHtrII forms a signaling complex with pharaonis phoborhodopsin (ppR) in the membrane, as also in the presence of a detergent. All tagged pHtrII showed a binding activity with ppR. Interestingly, the binding activity with ppR was positively correlated with the molecular weight of the soluble tags. Thus, larger soluble tags lead to higher binding activities. We could show, that our approach is beneficial for the preparation of active membrane proteins, and is also potentially applicable for larger membrane proteins, such as 7-transmembrane proteins.
- 著者
- Rei Abe Yoshizumi Shiori Kobayashi Mizuki Gohara Kokoro Hayashi Chojiro Kojima Seiji Kojima Yuki Sudo Yasuo Asami Michio Homma
- 出版者
- The Biophysical Society of Japan
- 雑誌
- BIOPHYSICS (ISSN:13492942)
- 巻号頁・発行日
- vol.9, pp.21-29, 2013 (Released:2013-02-05)
- 参考文献数
- 40
- 被引用文献数
- 2 2
Flagellar motors embedded in bacterial membranes are molecular machines powered by specific ion flows. Each motor is composed of a stator and a rotor and the interactions of those components are believed to generate the torque. Na+ influx through the PomA/PomB stator complex of Vibrio alginolyticus is coupled to torque generation and is speculated to trigger structural changes in the cytoplasmic domain of PomA that interacts with a rotor protein in the C-ring, FliG, to drive the rotation. In this study, we tried to overproduce the cytoplasmic loop of PomA (PomA-Loop), but it was insoluble. Thus, we made a fusion protein with a small soluble tag (GB1) which allowed us to express and characterize the recombinant protein. The structure of the PomA-Loop seems to be very elongated or has a loose tertiary structure. When the PomA-Loop protein was produced in E. coli, a slight dominant effect was observed on motility. We conclude that the cytoplasmic loop alone retains a certain function.