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    Cell-Free Synthesis of a Transmembrane Mechanosensitive Channel Protein into a Hybrid-Supported Lipid Bilayer

    Year: 2021

    Journal: ACS Appl. Bio Mater., Volume 4, APR 19, page 3101–3112

    Authors: Manzer, Zachary A.; Ghosh, Surajit; Jacobs, Miranda L.; Krishnan, Srinivasan; Zipfel, Warren R.; Pineros, Miguel; Kamat, Neha P.; Daniel, Susan

    Organizations: NSF [MCB-1935356, CBET-1844219]; Air Force Office of Scientific Research (AFOSR) [YIP FA9550-19-1-0039 P00001]; Searle Funds at The Chicago Community Trust; American Heart Association [20PRE35180215]; AFOSR [FA9550-19-1-0039 P00001]; National Institute of General Medical Sciences [T32GM008500]; Defense Advanced Research Projects Agency (DARPA) Army Research Office and accomplished [W911NF-18-2-0152]

    Keywords: supported lipid bilayer; cell-free protein synthesis; transmembrane proteins; lipids; diblock copolymers; hybrid vesicle

    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.
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