Emma Zhao discusses the crucial role of transmembrane protein expression in unveiling cellular functions and therapeutic opportunities.
The development of drugs targeting membrane proteins has caused a revolution in the field of pharmaceutical development. Approximately one-third of currently available drugs target G protein-coupled receptors (GPCRs); however, further development is limited due to the low expression levels, complex structures, instability during purification, in vitro insolubility, and difficulty in preserving the native conformation of these proteins. Overcoming these obstacles requires a combination of optimisation strategies, advanced purification techniques, and specialised expertise.
What are Transmembrane Proteins?
Membrane proteins constitute approximately one-third of all known sequence-encoded proteins, which fulfil vital roles in numerous cellular processes including molecular transport, energy utilisation, signal transduction, and maintenance of membrane homeostasis. Membrane proteins can be categorised into three types based on structure: peripheral, lipid anchored, or integral. Peripheral membrane proteins can be easily dissociated without disrupting the membrane structure, while lipid anchored membrane proteins are covalently attached to the bilayer via lipid moieties and can only be released by specific enzymes. Integral membrane proteins are firmly embedded within the lipid bilayer and must be removed by disrupting the membrane structure. These proteins serve various functions, including acting as G protein-coupled receptors (GPCRs), ion channels, transport proteins, and other types of membrane receptors. GPCRs are abundant in eukaryotes, where they interact with external molecules to initiate intracellular signaling, making them attractive targets for drug screening. Ion channels regulate the levels of ions such as sodium, potassium, and calcium; thus, playing a crucial role in maintaining cellular integrity. Transport proteins facilitate the movement of molecules and ions across biological membranes to regulate metabolic processes within organisms. Currently, about 60% of drug targets are membrane proteins.
Challenges for Membrane Protein Expression
Despite the huge potential of membrane proteins in drug development, recombinant expression of these proteins hinders the realisation of this potential for numerous reasons, as detailed below.
Membrane localisation. Transmembrane proteins are integrated into cellular membranes, which can make their expression more complicated compared to soluble proteins because ensuring proper targeting and insertion into the membrane is challenging, as is maintaining native conformation.
Hydrophobicity. Transmembrane proteins have large hydrophobic regions that span the lipid bilayer to anchor them in the membrane. This can cause problems for protein expression in aqueous environments, including misfolding, aggregation, or degradation.
Protein toxicity. Overexpression of transmembrane proteins, especially those with high hydrophobicity, can be toxic to the host cell; the accumulation of unfolded or misfolded proteins in the endoplasmic reticulum can activate cellular stress responses, such as the unfolded protein response, leading to cell death.
Membrane integration and folding. Transmembrane proteins require specific chaperones, translocation machinery, and other cellular factors for correct folding and membrane insertion. The absence or malfunction of these factors can result in inefficient membrane integration or misfolding of the protein.
Structural complexity. Transmembrane proteins often have complex structures with multiple domains, loops, and transmembrane segments. This complexity can hinder successful expression and purification, and complicate structural characterisation.
Low expression levels. Some transmembrane proteins have naturally low expression levels, which can make detection and purification challenging. Limited amounts of protein can hinder downstream functional and structural studies.
Three Multi-Pass Transmembrane Protein Development Platforms
Sino Biological has developed three platforms with advantages based on HEK293 expression, providing premium raw materials for early stage drug development. These platforms include virus-like particles (VLP), detergent micelles, and nanodiscs.
Virus-like Particle Platform
Sino Biological uses the VLP technology platform (based on the HEK293 mammalian expression system), which employs nano-scale, virus-derived self-assembling structures to facilitate the display of full length transmembrane target proteins on the VLP surface. This enables detection of expressed transmembrane proteins in their native state, which are then well-suited for immunisation and antibody screening purposes. Membrane proteins such as 4-pass (Claudin-6, Claudin-9, Claudin-18.1, Claudin-18.2) and 7-pass transmembrane proteins (GPRC5D, CXCR4, and SSTR2) have been found to have high activity levels after expression using this platform.
Detergent Micelle Platform
The detergent micelle technology platform ensures the production of membrane protein products optimised for immunisation, ELISA, SPR/BLI assays, and more by employing a conventional method of membrane protein extraction, in which surface-active compounds form non-covalent conformation-protected clusters in solution due to the hydrophobic effect.
Nanodisc Platform
The nanodisc platform takes advantage of the fact that transmembrane proteins maintain their functionality when attached to phospholipids to generate stable products, which are well-suited for applications such as immunization and antibody screening. The synthetic SMA polymer inserts into the cell membrane to “cookie-cut” the protein from the membrane while retaining the protein’s natural conformation and activity.
Featured GPRC5D Transmembrane Proteins
As a 7-pass transmembrane protein, GPRC5D has a very complex structure, which poses great challenges for antigen preparation, antibody development, and activity analysis. Sino Biological has successfully developed GPRC5D transmembrane protein products using three multi-pass transmembrane protein development platforms.
Emma Zhao is with Sino Biological.
