Cell-Free Membrane Protein Expression Services

Membrane Proteins

Membrane proteins are proteins that are embedded within or associated with biological membranes. They play crucial roles in the cell activities such as the transport of substances across the membrane and information exchange between the cell and the external environment. Membrane proteins participate in processes ranging from the nutrient uptake and the metabolite excretion to the transmission of neural signals and the recognition of the immune system.

As a component of the membrane, membrane proteins provide mechanical support to help maintain the shape of the cell. Due to the impermeability of the lipid bilayer of the cell membrane to most polar molecules and ions, membrane proteins are essential for the transmembrane transport of these substances. Many membrane proteins, such as the G protein-coupled receptor (GPCR) family can recognize and bind to specific extracellular signaling molecules, such as hormones, neurotransmitters, or growth factors. The binding triggers a series of biochemical reactions within the cell, transmitting external signals to the interior of the cell, thereby regulating various physiological processes such as cell growth, differentiation, and metabolism.

Cell-free Protein Expression Technology

Due to their hydrophobic domains, membrane proteins are difficult to fold and maintain activity in traditional cell-based expression systems. Cell-based expression systems often struggle with challenges such as low expression levels, the formation of inactive inclusion bodies, or toxicity to the host cells. Cell-free protein expression (CFPE) technology provides a promising alternative to overcome these challenges. The CFPE system mimics the protein synthesis environment within cells and contains all the core components required for transcription and translation with the DNA or mRNA template encoding the target protein to achieve rapid protein synthesis.

Advantages of CFPE Technology

• Efficient and rapid: By eliminating time-consuming steps such as cell culture, transformation, and selection, the target protein can be obtained within hours. This makes the CFPE technology an ideal choice for high-throughput screening and rapid functional validation.
• Open system and flexible regulation: The open reaction system allows the direct addition of various cofactors, inhibitors, unnatural amino acids, and stabilizing agents to the system, thereby optimizing the folding, stability, and function of membrane proteins.
• Expression of difficult-to-express proteins: Without the complex cellular activities and proteinases, the CFPE system is an ideal solution for the expression of those proteins that cause toxicity or are easily degraded in living cells.
• Simplified purification process: The components of the CFPE system are relatively simple and there is no interference from other cellular proteins, which makes the purification of the target protein more straightforward.
• High throughput and automation: The cell-free expression system is easy to miniaturize and automate, making it highly suitable for proteinomics research and drug screening and other fields that require large-scale parallel operations.

Cell-Free Membrane Protein Expression Services

Amerigo Scientific offers CFPE platforms based on detergents or nanodiscs for expressing membrane proteins. These CFPE systems can maintain the stability of membrane proteins, making the expression and purification easier compared to traditional expression methods. The success rate of expressing membrane proteins using our technology platform is nearly 100%. The membrane proteins that have been successfully expressed include cytochrome enzymes, mitochondrial respiratory chain enzymes, ligand receptors, etc.

Detergent-based Cell-Free Expression Platform

Utilizing our proprietary detergent-based CFPS technology, the soluble expression of full-length transmembrane proteins can be achieved. These added detergents provide a hydrophobic environment for the transmembrane domains of the membrane proteins, facilitating their proper folding into the native conformation.

Nanodisc-based Cell-Free Expression Platform

A nanodisc is a discoidal self-assembled complex composed of lipids and a helical protein known as membrane scaffold protein (MSP). By adding nanodiscs to the cell-free expression system, the synthesized membrane proteins directly incorporate into these nanodiscs. The hydrophobic transmembrane domains of membrane proteins are stably embedded in the lipid bilayer of nanodiscs. The membrane proteins expressed by the nanodisc-based CFPE system exhibit enhanced stability, functional activity, and conformations similar to the natural ones.

Technical Advantages

• Optimized expression systems according to the characteristics of different proteins
• High purity (~80%), the low endotoxin level (1EU/ug)
• Efficient expression process, with short delivery time

Technical Procedures

Deliverables

• Target plasmid (2 ug)
• A report for protein expression and purification
• Target membrane protein (purity of ~80%, the lowest endotoxin of 1EU/ug)

Application Cases of Nanodisc-based Cell-free Expression

Application Cases of Detergent-based Cell-free Expression