Take Aways
Abstract
Membrane proteins represent a class of proteins that are difficult targets to characterize. Their structural and functional characterization requires that they first be produced at quantities that enable their biophysical and biochemical analysis. Because they are natively produced at levels much lower than their soluble counterparts, extraction from their natural sources is not sufficient to produce enough material for these studies. Recombinant protein expression and production has become a popular method to produce large amounts of proteins for research and industrial purposes. Significant effort has been spent finding new ways to optimize and increase protein expression. As cutting edge techniques in synthetic biology continue to advance they offer a potential well of opportunities to tune expression through better control of the transcription and translation processes. Many techniques being developed are geared toward the production of soluble proteins, but in the following review, a focus on effective strategies to maximize membrane protein production in yeast is presented and includes many of the most innovative approaches to maximize expression using synthetic biology. Synthetic biology utilizes modern techniques in molecular biology and genetic engineering to optimize the production of compounds produced in microbes by altering gene elements required for transcription and translation of critical genes responsible for their synthesis. Compounds include natural products, hydrocarbon-based compounds for biofuels, and therapeutic proteins. Producing membrane proteins recombinantly using similar methods to increase expression yields is described in this review along with cutting edge techniques like cell-free expression, which circumvents many of the common problems that plague overexpression of membrane proteins microbial-based platforms.
Introduction
Membrane proteins (MPs) represent an important class of biomolecules that either closely associate with or almost completely reside within the membranes of cells. They are crucially important in cellular processes ranging from signaling, trafficking, and more recently, scaffolding and shaping of the plasma membrane. Regions of exposed hydrophobic amino acid residues form intimate contacts with membranes that help to stabilize their structure and function (Levental & Lyman, 2022). They also render these proteins remarkably challenging to study. Many biochemical and biophysical techniques used in the preparation of their soluble counterparts must often be adapted through the addition of detergents and lipid mimetic complexes that provide a membrane-like environment. One major obstacle to their study is producing quantities of biologically active proteins for structural characterization and other downstream analyses.
MPs can be divided into three broad classes: peripheral, integral, and lipid-anchored. Peripheral MPs interact with the plasma membrane superficially wherein only a small portion or region of exposed hydrophobic amino acid residues are in contact with the lipid bilayer. These proteins can typically be extracted using biochemical techniques suitable for soluble proteins and do not necessitate the addition of detergents or lipids to increase their solubility in aqueous buffers. Similarly, lipid-anchored proteins are mostly soluble in aqueous buffers and rely on the covalent attachment of a lipid (e.g. palmitoylation) or a glycolipid (e.g. glycophosphatidylinositol) to one or more residues to interact with membranes. Integral membrane proteins (IMPs), which will be primarily referred to in this article, are almost entirely, > 50% amino acid composition, embedded in the lipid bilayer of the plasma membrane of cells rendering them extremely insoluble. In vitro , a suitable detergent or lipid complex must be used to keep them soluble and functionally active (Czerski & Sanders, 2000; Levental & Lyman, 2022; Lin & Guidotti, 2009; Whiles et al., 2002). Methods used to obtain proteinaceous material for in vitro analyses can rarely be universally applied across the entire spectrum of IMPs. Efforts to optimize experimental conditions is resource and labor-intensive and hampers progress toward characterization. As a result, relatively few IMPs have known solved structures compared to soluble proteins (Carpenter et al., 2008; Pan & Vachet, 2022). G-protein coupled receptors (GPCRs), for example, are one of the largest classes of IMPs (Errey & Fiez-Vandal, 2020). They are a key player in signal transduction and are responsible for processing extracellular signals across cell membranes leading to a downstream response. They are nearly ubiquitous across all kingdoms of life, but their importance in critical cellular processes, specifically in humans, makes them popular targets for drug therapy. Elucidating the structure of GPCRs has direct implications for rational drug design. Until 2007, the three-dimensional structure of nearly all GPCRs remained uncharacterized (Cherezov et al., 2010; Qu et al., 2020). Fortunately, critical advances in experimental methods such as the advent of cryo-electron microscopy (cryo-EM) have enabled significant achievements to be made. Although, high-yield production and purification still remains a formidable challenge to their understanding and behaviorin vivo .
Synthetic biology is a field uniquely poised to address the expression problem in membrane protein research. In this review, techniques used in the field of synthetic biology are explored presenting potentially the most effective ways to fine-tune expression and production to maximize yields. Recombinant expression methods serve as the basis for this discussion to provide a background for understanding the underlying challenges associated with current methods in MP expression. Along these lines, the molecular biology steps that govern critical intracellular processes in yeast is described to highlight important areas that might be targeted to address some of the most difficult challenges. Transcription and translation are two processes that lie at the center of protein expression and production. They affect intracellular conditions in yeast that ultimately effect cell viability and final yields. Many of the synthetic biology strategies discussed take into account regulatory features involved in transcription such as choice of gene promoters, terminators and other genetic elements that provide a greater level of control, which affects the viability of yeast during expression as well as the quantity and potential quality of the protein produced.