Take Aways
- Membrane proteins are challenging targets for recombinant expression
- Overexpression results in misfolding, aggregation, degradation and
apoptosis
- Synthetic biology helps tune protein expression and increase yields
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.