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

Abstract

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.

Expression of Fatty Acyl-CoA Ligase Drives One-Pot De Novo Synthesis of Membrane-Bound Vesicles in a Cell-Free Transcription-Translation System

“…The well-defined nature of the PURE system makes it suitable for applications requiring the generation of components and building blocks for synthetic cells . The PURE system is compatible with various preformed lipidic structures such as liposomes, nanodiscs, sponge droplets, and supported lipid bilayers . However, such preformed structures are passive components and are unlikely to directly interfere with the chemistry of the PURE system.…”

Expression of fatty acyl-CoA ligase drives one-pot de novo synthesis of membrane-bound vesicles in a cell free transcription-translation system

“…2 The PURE system is compatible with various pre-formed lipidic structures such as liposomes, 22 nanodiscs, 23 sponge droplets, 24 and supported lipid bilayers. 25 However, such pre-formed structures are passive components and are unlikely to directly interfere with the chemistry of the PURE system. A more challenging goal is to integrate active lipid synthesis pathways into the PURE system.…”

Synthetic Cell as a Platform for Understanding Membrane-Membrane Interactions

“…αHL has been reconstituted using different bilayer platforms, such as liposomes [76,77], SLBs [78,79], and DIBs [39]. The ease of αHL self-assembly in the membrane enables a variety of applications that require transporting ions and molecules across the membrane, including biosensing [39], coacervation [80], and activation of genetic circuits [11]. For example, Adamala et al generated liposomes encapsulating genetic circuits and cell lysates with transcriptional and translational activity and used αHL to enable membrane permeability of small molecular inducers (Figure 2A) [81].…”

“…Attempts to reconstitute MscL have been made in various types of planer lipid bilayers, including DHBs, SLBs, and DIBs [11,[90][91][92] [91,92]. It has only been in recent years that MscL was used in synthetic cells.…”

“…Many platforms are available where lipid bilayers are created, including standing bilayer membrane, supported bilayer, and unilamellar lipid vesicles (see Section 2). Standing bilayer is utilized for the study of ion channels and membrane proteins [8], while supported bilayer is utilized for membrane fusion [9] and protein expression [10,11]. Micron-sized unilamellar vesicles are used as a synthetic cell model for studying biochemical reactions in vitro [7,12].…”