Cell-Free Synthesis of a Transmembrane Mechanosensitive Channel Protein into a Hybrid-Supported Lipid Bilayer
Cell-Free Synthesis of a Transmembrane Mechanosensitive Channel Protein into a Hybrid-Supported Lipid Bilayer
复制标题
将跨膜机械敏感通道蛋白无细胞合成到混合支持的脂质双层中
DOI：
10.1021/acsabm.0c01482
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发表时间：
2021
影响因子：
4.7
通讯作者：
Daniel, Susan
中科院分区：
文献类型：
--
作者：
Manzer, Zachary A.;Ghosh, Surajit;Jacobs, Miranda L.;Krishnan, Srinivasan;Zipfel, Warren R.;Piñeros, Miguel;Kamat, Neha P.;Daniel, Susan
支持的脂质双层(SLB)作为细胞模拟结构具有巨大的前景，可以很容易地与分析和筛选工具相结合。生物膜的关键成分跨膜蛋白的掺入是一个重大挑战，限制了SLB用于各种生物技术应用的能力。在这里，我们报告了一种使用无细胞表达系统将膜蛋白共翻译插入含有磷脂和两嵌段共聚物的杂化支持的脂质双层(HSLB)中的方法。我们使用无细胞表达技术和一个模型跨膜蛋白，大电导机械敏感通道(MSCL)，展示了两种途径将通道蛋白整合到HSLB中。我们发现，HSLB可以通过将蛋白质共翻译整合到杂合小泡中，然后融合这些蛋白脂质体形成HSLB，或者预先形成HSLB，然后直接将蛋白质无细胞合成到HSLB中，从而与整合的膜蛋白组装。这两种方法都会导致HSLB与定向蛋白的组装。值得注意的是，使用单粒子跟踪，我们发现两嵌段共聚物的存在促进了HSLB中膜蛋白的流动性，这是一个在纯脂类SLB中难以实现的关键特征。本文提出的利用无细胞共翻译插入将膜蛋白直接整合到预制的HSLB中的方法是实现许多生物技术应用的重要一步，包括生物传感、药物筛选和需要细胞膜样界面的材料平台，这些界面将非生物和生物世界结合在一起，并依赖跨膜蛋白作为转导元件。
Supported lipid bilayers (SLBs) hold tremendous promise as cellular-mimetic structures that can be readily interfaced with analytical and screening tools. The incorporation of transmembrane proteins, a key component in biological membranes, is a significant challenge that has limited the capacity of SLBs to be used for a variety of biotechnological applications. Here, we report an approach using a cell-free expression system for the cotranslational insertion of membrane proteins into hybrid-supported lipid bilayers (HSLBs) containing phospholipids and diblock copolymers. We use cell-free expression techniques and a model transmembrane protein, the large conductance mechanosensitive channel (MscL), to demonstrate two routes to integrate a channel protein into a HSLB. We show that HSLBs can be assembled with integrated membrane proteins by either cotranslational integration of protein into hybrid vesicles, followed by fusion of these proteoliposomes to form a HSLB, or preformation of a HSLB followed by the cell-free synthesis of the protein directly into the HSLB. Both approaches lead to the assembly of HSLBs with oriented proteins. Notably, using single-particle tracking, we find that the presence of diblock copolymers facilitates membrane protein mobility in the HSLBs, a critical feature that has been difficult to achieve in pure lipid SLBs. The approach presented here to integrate membrane proteins directly into preformed HSLBs using cell-free cotranslational insertion is an important step toward enabling many biotechnology applications, including biosensing, drug screening, and material platforms requiring cell membrane-like interfaces that bring together the abiotic and biotic worlds and rely on transmembrane proteins as transduction elements.