Fraunhofer Institute for Cell Therapy and Immunology (IZI), Branch Bioanalytics and Bioprocesses (IZI-BB), Am Mühlenberg 13, 14476 Potsdam, Germany; Institut für Biochemie und Biologie, University of Potsdam, Karl-Liebknecht-Str. 24-25, 14476 Potsdam OT Golm, Germany.
Fraunhofer Institute for Cell Therapy and Immunology (IZI), Branch Bioanalytics and Bioprocesses (IZI-BB), Am Mühlenberg 13, 14476 Potsdam, Germany.
Biochim Biophys Acta Biomembr. 2023 Jun;1865(5):184144. doi: 10.1016/j.bbamem.2023.184144. Epub 2023 Mar 6.
Here, we demonstrate the utility of native membrane derived vesicles (nMVs) as tools for expeditious electrophysiological analysis of membrane proteins. We used a cell-free (CF) and a cell-based (CB) approach for preparing protein-enriched nMVs. We utilized the Chinese Hamster Ovary (CHO) lysate-based cell-free protein synthesis (CFPS) system to enrich ER-derived microsomes in the lysate with the primary human cardiac voltage-gated sodium channel 1.5 (hNa1.5; SCN5A) in 3 h. Subsequently, CB-nMVs were isolated from fractions of nitrogen-cavitated CHO cells overexpressing the hNa1.5. In an integrative approach, nMVs were micro-transplanted into Xenopus laevis oocytes. CB-nMVs expressed native lidocaine-sensitive hNa1.5 currents within 24 h; CF-nMVs did not elicit any response. Both the CB- and CF-nMV preparations evoked single-channel activity on the planar lipid bilayer while retaining sensitivity to lidocaine application. Our findings suggest a high usability of the quick-synthesis CF-nMVs and maintenance-free CB-nMVs as ready-to-use tools for in-vitro analysis of electrogenic membrane proteins and large, voltage-gated ion channels.
在这里，我们展示了天然膜衍生囊泡（nMVs）作为快速进行膜蛋白电生理分析工具的效用。我们使用无细胞（CF）和基于细胞（CB）的方法来制备富含蛋白质的 nMVs。我们利用基于中国仓鼠卵巢（CHO）裂解物的无细胞蛋白合成（CFPS）系统，在 3 小时内使 ER 衍生的微体在裂解物中富集主要来源于人类心脏电压门控钠离子通道 1.5（hNa1.5；SCN5A）。随后，从过表达 hNa1.5 的氮空化 CHO 细胞的级分中分离出 CB-nMVs。在综合方法中，nMVs 被微移植到非洲爪蟾卵母细胞中。CB-nMVs 在 24 小时内表达出天然的利多卡因敏感的 hNa1.5 电流；CF-nMVs 则没有引起任何反应。CB-nMV 和 CF-nMV 制剂都在平面脂质双层上引发单通道活性，同时保持对利多卡因应用的敏感性。我们的研究结果表明，快速合成的 CF-nMVs 和无需维护的 CB-nMVs 可作为即用型工具，用于电活性膜蛋白和大型电压门控离子通道的体外分析。
