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Technological Breakthrough: Perrotin Achieves Cell-Free Protein Expression Combined with Nanodiscs for Ultra-High-Efficiency Expression of Full-Length Membrane Proteins
Release date:
2024-11-21
A new breakthrough has been achieved in the Perotin biomembrane protein expression platform!
Recently, Perotin Bio has achieved the highly efficient direct incorporation of membrane proteins into nanodiscs by incorporating nanodiscs into its proprietary cell-free protein synthesis (CFPS) system.
Perotin possesses a full suite of proprietary intellectual property!
This technology is expected to significantly enhance the overall efficiency of membrane protein research and development in China.
A New Option for Membrane Protein Expression
Membrane proteins play crucial roles in numerous cellular processes, such as transmembrane transport and signal transduction. Due to the presence of hydrophobic transmembrane domains (TMDs), recombinant expression of membrane proteins often faces challenges, including low yields and conformational instability.
Cell-free protein synthesis (CFPS) is an in vitro biochemical reaction that directly produces proteins. This technology isolates the molecular machinery required for intracellular protein synthesis—such as RNA polymerase, ribosomes, and transcription‑translation cofactors—and supplements it with nucleotides, amino acids, energy substrates, and a genetic template, enabling protein synthesis outside living cells.
CFPS offers high controllability and flexibility, allowing detergents or nanodiscs to be directly incorporated into the system to provide a membrane-like environment for membrane proteins, thereby enabling soluble expression of full-length membrane proteins.
Figure 1. Expression of membrane proteins in a cell-free system [1]
A New Breakthrough in the Assembly of Cell-Free Expression–Coupled Nanodiscs
Recently, Perotin Bio incorporated Nanodiscs into its proprietary cell-free system, successfully expressing a membrane protein fused with green fluorescent protein (GFP) and demonstrating that this membrane protein was efficiently incorporated into the nanodiscs. As shown in Figure 2, the membrane protein precipitated and was expressed in a CFPS system without detergents; however, soluble expression was achieved upon addition of either a surfactant or Nanodiscs, with the Nanodisc‑containing condition yielding superior expression compared to the detergent‑only group.
Figure 2: Expression of CFPS+Nanodisc
The membrane scaffold protein (MSP) used in this experiment carries a His‑tag for purification, whereas the GFP‑fused membrane protein lacks a His‑tag. As shown in Figure 3, following nickel‑affinity purification, the gel clearly reveals a distinct band corresponding to the target membrane protein, indicating that the target protein was successfully purified via the MSP’s His‑tag and incorporated into the nanodiscs.
Figure 3: Assembly of CFPS+Nanodisc
The “cradle” of membrane proteins
Nanodiscs are disc-shaped, membrane-like structures composed of membrane scaffold proteins (MSPs) and phospholipid molecules, capable of providing a stable, near-native membrane environment for membrane proteins.
Figure 4 Schematic diagram of the nanodisc structure [2]
The significant advantages of the nanodisc system are:
① Nanodiscs can provide a phospholipid bilayer environment that more closely resembles the native cell membrane, thereby facilitating the formation of correct native conformations of membrane proteins.
②Nanodiscs are more conducive to maintaining the conformational stability of membrane proteins, thereby facilitating downstream purification.
③ Membrane proteins reconstituted in nanodiscs are more amenable to downstream analyses, can be directly subjected to ELISA assays, and are suitable for structural biology applications, including NMR, EPR, and other techniques.
④ By flexibly employing membrane scaffold proteins of varying lengths, Nanodiscs can accommodate membrane proteins of different sizes.
Cell-free systems enable the simultaneous expression and incorporation of membrane proteins into nanodiscs. Perotin Bio has achieved direct assembly of expressed membrane proteins into nanodiscs by adding nanodiscs directly to the cell-free reaction mixture. Compared with cell-based nanodisc platforms, this cell-free expression–nanodisc assembly approach eliminates the need for a separate detergent‑solubilization step after expression, thereby minimizing perturbations to the protein’s conformation and significantly simplifying membrane protein purification.
From now on, Chinese life scientists will no longer face bottlenecks in achieving 100% expression of membrane proteins!
References
[1] Manzer ZA, Selivanovitch E, Ostwalt AR, Daniel S. Membrane protein synthesis: no cells required. Trends Biochem Sci. 2023 Jul;48(7):642-654. doi: 10.1016/j.tibs.2023.03.006. Epub 2023 Apr 20. PMID: 37087310.
[2] Gulezian E, Crivello C, Bednenko J, Zafra C, Zhang Y, Colussi P, Hussain S. Membrane protein production and formulation for drug discovery. Trends Pharmacol Sci. 2021 Aug;42(8):657-674. doi: 10.1016/j.tips.2021.05.006. PMID: 34270922.
Perotin Bio was founded in 2017 in the Suzhou Industrial Park and is a specialized biotechnology company focused on cell-free protein expression. The company boasts a highly skilled technical team comprising national-level leading talents and overseas‑returned PhDs. Leveraging its independently developed, uniquely distinctive cell‑free protein expression technology platform, Perotin Bio provides cell‑free protein expression products, protein‑based reagents, and customized services to a wide range of biopharmaceutical and synthetic biology companies and research institutions.
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Phone: 0512-67900128
Official website: www.cellfreeprotein.cn
Address: Room 302, Building 1, Weili Medical Science and Technology Park, No. 69 Jiepu Road, Suzhou Industrial Park
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