ABSTRACT
There aren’t supposed to be intact cell walls or membranes remaining in in vitro expression systems, so one can think about it as an “open system,” which allows the addition of components, such as labeled amino acids or nonnatural amino acids in the form of synthetic tRNAs [16] to enter the ribosomal protein factory-thus, one can label the protein of interest by adding labeled building blocks, such as fluorescent, spin-label, or isotopic groups. Besides, no elaborate setup is required for the synthesis, and depending on the following analysis method, there is no need for further purification steps. In certain optimal examples, a competitive, large amount of purified protein (several mg/mL) can be generated in a few hours’ time using batch reactions [17], the continuous-flow cell-free (CFCF) [18] method, or the semi-continuous-exchange cell-free (CECF) method [19, 20]. The CECF method elongates the reaction life time due to a continuous exchange of substrates and by-products through a dialysis membrane or a lipid bilayer [19]. A common strategy to avoid some of the problems associated with conventional expression difficulties, the purification process, and functioning of membrane proteins is to express the protein of interest by using a cell-free system. CFPS is known to offer a rapid and high-throughput expression strategy and increased production for soluble proteins. It also gives an opportunity to work with the proteins that are difficult to handle, like toxic and insoluble proteins. This topic is our focus: exploring CFPS for membrane protein expression. One of the potential and comprehensive impacts of in vitro expression could be in the future area of membrane protein production. Membrane proteins account for nearly a third of the genes encoded by most fully sequenced genomes. However, only a few examples of integral membrane protein structures (~250, https://blanco.biomol.uci.edu/Membrane_ Proteins_xtal.html) have been observed and characterized because of the overexpression limitations. Overexpression of membrane proteins in vivo frequently results in cell death, largely owing to their hydrophobicity, protein aggregation, and misfolding-in some cases, one might think of apoptosis induction. In some cases, the overexpression of integral membrane proteins such as ion channel proteins, transporters, and receptors can disrupt the integrity of the cell membrane and lead to cell lysis. We like to challenge these intrinsic difficulties by a “bypass” to the cellular context: the CFPS strategies. The most important requirements for the expression
of membrane proteins is the availability of appropriate lipophilic matrices provided by detergent micelles, lipid membranes, microsomes, nanolipoprotein particles, or liposomes. Several studies have described the functional expression of membrane proteins by the cell-free method with optimized detergents and lipid compositions in solution [21-26]. Our strategy differs in a way that we immobilize the lipid bilayer membranes on a surface appropriate for real-time analysis. As an example for the potential of CFPS to “handle” membrane proteins, we introduce the terminal supramolecular complex from the respiratory chain. The respiratory chain, also known as the electron transport chain, is a series of four-membrane bound protein complexes; each one comprised of a multisubunit enzyme complex that is integrated in the inner membrane of mitochondria in eukaryotes and in the plasma membrane of prokaryotes. All members of this chain contribute to adenosine triphosphate (ATP) synthesis by controlling proton and electron flow through the membranes. Yet we address in here one component of this chain-cytochrome bo3ubiquinol oxidase (Cyt-bo3), which is the bacterial analogue of the mammalian cytochrome c oxidase [27, 28]. Cyt-bo3 is an important member of heme-copper terminal oxidases. The E. coli Cyt-bo3complex is a four-subunit enzyme complex, which contains two heme (heme b and heme o) prosthetic groups and one copper atom (CuB) in its active center. In total it contains 25 transmembrane-spanning domains and a large C-terminal hydrophilic domain in the periplasm [29, 30]. A major example of difficult cases of CPFS is to synthesize fully functional Cyt-bo3 ubiquinol oxidase on an artificial membrane surface by maintaining its structural integrity as its natural form. In previous years, we have introduced a platform for membrane protein integration, which mimics the amphiphilic architecture of a cell-derived membrane system [21]. Research about membrane proteins is facing severe obstacles; therefore still just a few examples of membrane protein species have been characterized in suitable experimental platforms [31, 32]. The mimic of a biological membranes such as solid-supported lipid membranes is widely used as a platform to investigate protein membrane interactions [33, 34]. A spacer molecule separates the planar lipid system from the substrate and provides a reservoir place for insertion of the protein. The inner leaflet of the tethered biomolecular membrane (tBLM) is covalently
coupled to the solid support via a spacer group. Such systems have been shown to provide excellent stability and electrical properties, as well as high efficiency to incorporate membrane proteins, which is satisfactory for biosensing applications. So this membrane system creates a suitable environment for in vitro expression of complex transmembrane proteins [35], such as Cyt-bo3 ubiquinol oxidase. As a well-established architecture, we like to mimic a biological membrane on a planar solid support, namely, tBLM. This artificial membrane system consists of a hydrophilic spacer molecule (P19, α-laminin peptide), which provides an aqueous reservoir for the integration of outer domains of the membrane protein, as a monolayer. 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE) layer was chosen as the top layer of the peptide spacer. The DMPE layer was completed into a biomembrane-like bilayer by an L-α-Phosphatidylcholine (PC) (from soybean) molecule, resulting from a vesicle fusion procedure. This membrane architecture can be characterized by using surface plasmon resonance spectroscopy (SPR). SPR has been widely used as a detection technique in biosensor applications as well as for characterizing molecular interactions at the interface between analytes and a sensor surface. This technique is described in detail elsewhere in this book, since this technique is perfectly suited to characterize thin biofilms and biomolecular interaction in general. The SPR method offers a label-free, mechanically noninvasive and real-time detection method. Surface plasmon-enhanced fluorescence spectroscopy (SPFS) was introduced as an extension of SPR, which improves the sensitivity of SPR by combining it with the detection principles of fluorescence. SPFS uses the enhanced electromagnetic field obtained by SPR to excite fluorescent dyes in close proximity to the metal/dielectric interface. Furthermore, SPFS gives an opportunity to detect specific interactions in between the aimed protein and the corresponding antibody [36-38]. Taken together, artificial membrane technology, combined with in vitro synthesis of membrane proteins results in interesting architectures to be characterized by various methods. The interesting strategy is explained in Fig. 19.1, which consists of tBLM formation followed by the CFPS process, namely, in vitro expression of Cyt-bo3 in an artificial membrane system. Protein insertion and orientation is detected via the SPFS method by using specific interaction of the protein-antibody sandwich system, as shown within the same figure.
