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Cell-free expression opens new avenues for studying membrane protein function.
Release date:
2026-07-09
Membrane proteins make up 30% of all proteins encoded by the human genome and account for over 60% of all drug targets [1]. They are widely distributed across cell membranes and organelles, acting as central regulators of biological activities. Because the dysregulation of membrane proteins can lead to a variety of diseases, they have become highly sought-after targets for drug development [2].
I. Key Functions of Membrane Proteins
Substance Transport: Including ion channels, transport proteins, and the transmembrane transport of nutrients, membrane proteins provide essential mechanisms for maintaining cellular homeostasis.
Signal Transduction: Receptor proteins (like GPCRs) are responsible for receiving extracellular signals and transmitting them intracellularly. GPCRs represent the largest proportion of approved drug targets [1-2].
Cellular Recognition and Adhesion: Membrane proteins mediate immune recognition, intercellular junctions, and tissue distribution [1].
Enzymatic Activity and Structural Support: Membrane proteins inherently possess enzymatic functions while also helping maintain cytoskeletal connections and the stability of membrane structures [4].
Figure 1: Seven-transmembrane (7TM) receptor signal transduction. Nat Rev Mol Cell Biol. 2002
II. Challenges in Membrane Protein Synthesis
Membrane proteins are widely considered the most difficult class of proteins to express due to their hydrophobicity, multiple transmembrane domains, and folding complexity. Resolving their structures remains a core, long-standing challenge in structural biology.
Low Success Rates in Cellular Systems: Because transmembrane domains are highly hydrophobic, proteins easily misfold or aggregate inside cells. Furthermore, many membrane proteins are toxic to cells, directly leading to host cell death [5]. Consequently, achieving highly efficient expression for most membrane proteins in cell-based systems is incredibly difficult.
Proper Conformation Relies on the Phospholipid Bilayer: Most membrane proteins require a native membrane structure to fold correctly, but traditional cell expression systems struggle to accurately provide these highly specific environments [6].
Difficult Detergent Screening and High Purification Costs: Different membrane proteins have unique detergent preferences, demanding extensive experimental screening. Additionally, the folded state is easily disrupted during the purification process [5].
Poor Stability and Low Purity: The poor stability and high tendency to aggregate of membrane proteins represent some of the biggest bottlenecks in structural research.
III. Cell-Free Protein Synthesis (CFPS): A Breakthrough for Membrane Protein Research
Cell-Free Protein Synthesis (CFPS) enables the direct synthesis of proteins in an in vitro environment. Characterized by its open nature and lack of toxicity limitations, CFPS is recognized as a key technology for overcoming the hurdles of membrane protein expression.
Zero Cytotoxicity: CFPS doesn't involve an intracellular environment, so it's completely unaffected by cellular toxicity. This makes it possible to express channel proteins or transmembrane proteins that would otherwise be toxic to host cells.
Compatible with Detergents and Nanodiscs for Assisted Folding: The system can synthesize folding environments that closely mimic native membranes, significantly boosting the expression yield, success rate, and functional activity of membrane proteins.
Highly Controllable Open System: It offers high flexibility and precise control over the reaction environment.
Enables Rapid Construction and Screening: It is perfectly suited for mutant screening and high-throughput protein function screening platforms.
Figure 2: Flowchart comparing cell-free systems with traditional in vivo systems
IV. PLD Technology's Cell-Free Protein Expression System Empowers Breakthroughs in Membrane Protein Research
Building on our proprietary cell-free protein expression technology, PLD Technology has developed a comprehensive product portfolio ranging from standalone kits to one-stop membrane protein expression services. By introducing folding cofactors like nanodiscs and detergents directly into the expression reaction, we can effectively enhance the solubility and functional acquisition of notoriously hard-to-express membrane proteins, such as multi-transmembrane receptors, transporters, and ion channels. We have successfully achieved structural-research-grade expression yields and activity across a variety of membrane protein projects, significantly shortening the R&D timeline for our scientific clients from "gene to functional protein."
Looking ahead, with the continuous iteration of technologies like nanodisc-assisted folding, non-natural amino acid labeling, and automated high-throughput screening, PLD Technology's cell-free platform will further expand its application scope in membrane protein structural biology, antibody drug discovery, and novel target validation. We are committed to providing scientific research and drug development with membrane protein solutions that offer higher success rates, shorter timelines, and greater certainty.
References
[1] Overington JP, Al-Lazikani B, Hopkins AL. How many drug targets are there?. Nat Rev Drug Discov. 2006;5(12):993-996.
[2] Venkatakrishnan AJ, Deupi X, Lebon G, Tate CG, Schertler GF, Babu MM. Molecular signatures of G-protein-coupled receptors. Nature. 2013;494(7436):185-194.
[3] Pierce KL, Premont RT, Lefkowitz RJ. Seven-transmembrane receptors. Nat Rev Mol Cell Biol. 2002;3(9):639-650.
[4] White SH, Wimley WC. Membrane protein folding and stability: physical principles. Annu Rev Biophys Biomol Struct. 1999;28:319-365.
[5] Seddon AM, Curnow P, Booth PJ. Membrane proteins, lipids and detergents: not just a soap opera. Biochim Biophys Acta. 2004;1666(1-2):105-117.
[6] Corradi V, Mendez-Villuendas E, Ingólfsson HI, et al. Lipid-Protein Interactions Are Unique Fingerprints for Membrane Proteins. ACS Cent Sci. 2018;4(6):709-717.
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