Concurrent In Vitro Synthesis and Functional Detection of Nascent Activity of the KcsA Channel under a Membrane Potential,ACS Synthetic Biology

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Concurrent In Vitro Synthesis and Functional Detection of Nascent Activity of the KcsA Channel under a Membrane Potential
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2018-03-22 00:00:00 , DOI: 10.1021/acssynbio.7b00454
Masayuki Iwamoto ₁ , Maie A. Elfaramawy ₂ , Mariko Yamatake ₁ , Tomoaki Matsuura ₂ , Shigetoshi Oiki ₁

Affiliation

Processes involved in the functional formation of prokaryotic membrane proteins have remained elusive. Here, we developed a new in vitro membrane protein expression system to detect nascent activities of the KcsA potassium channel in lipid bilayers under an applied membrane potential. The channel was synthesized using a reconstituted Escherichia coli-based in vitro transcription/translation system (IVTT) in a water-in-oil droplet lined by a membrane. The synthesized channels spontaneously incorporated into the membrane even without the translocon machinery (unassisted pathway) and formed functional channels with the correct orientation. The single-channel current of the first appearing nascent channel was captured, followed by the subsequent appearance of multiple channels. Notably, the first appearance time shortened substantially as the membrane potential was hyperpolarized. Under a steadily applied membrane potential, this system serves as a production line of membrane proteins via the unassisted pathway, mimicking the bacterial synthetic membrane.

中文翻译：

膜电位下同时体外合成和功能检测KcsA通道的新生活性。

涉及原核膜蛋白功能形成的过程仍然难以捉摸。在这里，我们开发了一种新的体外膜蛋白表达系统，以检测在施加的膜电位下脂质双层中KcsA钾通道的新生活性。使用重组的基于大肠杆菌的体外合成通道膜衬里的油包水液滴中的转录/翻译系统（IVTT）。合成的通道即使没有translocon机器（无辅助通道），也会自发地并入膜中，并形成具有正确方向的功能性通道。捕获第一个出现的新生通道的单通道电流，然后随后出现多个通道。值得注意的是，随着膜电位超极化，首次出现时间大大缩短。在稳定施加的膜电位下，该系统通过无辅助途径充当膜蛋白的生产线，模仿细菌合成膜。

更新日期：2018-03-22

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