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

• Published in: ACS applied bio materials Vol. 4; no. 4; pp. 3101 - 3112
• Main Authors: Manzer, Zachary A, Ghosh, Surajit, Jacobs, Miranda L, Krishnan, Srinivasan, Zipfel, Warren R, Piñeros, Miguel, Kamat, Neha P, Daniel, Susan
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 19.04.2021
• Subjects: Biocompatible Materials - chemistry; Cell-Free System - chemistry; Lipid Bilayers - chemistry; Materials Testing; Membrane Proteins - chemistry; Particle Size; transmembrane proteins; lipids; cell-free protein synthesis; hybrid vesicle; supported lipid bilayer; diblock copolymers
• ISSN: 2576-6422; 2576-6422
• DOI: 10.1021/acsabm.0c01482

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.