Membrane proteins are key biological players involved in myriad essential cellular processes from molecular transport, cellular communication, and signal transduction to maintaining structures within cells and tissues. Despite their importance, accounting for 25% of all proteins in the human genome and two-thirds of druggable protein targets (1), they are significantly less documented compared to soluble proteins, with less structural information available, such that data at atomic resolution is more difficult to obtain.
As membrane proteins are often embedded into phospholipid bilayers, when trying to extract them in the lab it is essential to be able to disrupt the membrane without affecting the protein’s structure. Additional challenges relate to difficulty in solubilizing membrane proteins and ensuring that they remain stable and functional (2). This article will review a few essential tips that should be borne in mind when expressing, extracting, and purifying membrane proteins, to maximize the chances of success.
The expression challenge
“Membrane proteins typically express quite poorly compared to the amounts you might expect of soluble proteins, so this immediately creates challenges in downstream studies requiring large amounts or high concentrations of membrane protein,” says Chris Beckwith PhD, ÄKTA & Chromatography Specialist at Cytiva.
In terms of expression systems, Escherichia coli is one of the most frequently used for membrane proteins. Unfortunately, when membrane proteins are over-expressed, they can cause toxicity issues, as well as issues with achieving correct post-translational modifications, folding, and accurate insertion into membranes. Although fusing green fluorescent protein to the gene of interest is commonly used as a quality control indicator, using fluorescence levels as a readout of proteins correctly inserted into membranes might be misleading. Recent research has demonstrated that this measure might not accurately indicate the membrane protein is properly folded.
It is also important to bear in mind that the E. coli strain used can influence the functionality of individual membrane proteins. This should be considered when selecting a strain, rather than focusing solely on a strain’s level of protein expression (3). Another effective strategy, if membrane availability for accurate insertion is a potential issue, is to use another species of bacteria that has more internal membrane available such as Rhodobacter (4), or to alternatively produce the protein as inclusion bodies (5).
With regard to difficulties isolating membrane proteins and maintaining intrinsic function, protein engineering can help mitigate some of these issues, to alter the physical properties of the membrane protein to facilitate structural analysis, including the development of membrane protein mutant libraries using rational design, random mutagenesis, scanning mutagenesis, and consensus mutation (1).
Considerations for membrane protein extraction
Beckwith says structural studies using x-ray crystallography provide a good example of the challenge that membrane proteins can present during extraction and reconstitution. “Membrane proteins are often solubilized from the membrane fraction or inclusion bodies of a cell lysate using detergents, meaning that a wide range of detergents and detergent concentrations need to be tested to see which one will provide a more stable or more active reconstituted protein.”
The complexity added by this need for additional screening means that it is generally more expensive when compared to soluble protein targets, Beckwith adds. The use of detergents also creates sample polydispersity or non-uniformity of distribution, which can negatively affect experimental outcomes especially in structural studies.
Buffer selection is critical, and often Tris buffers (pH 6.5–9.5), HEPES or phosphate buffers are selected to help maintain conformational integrity as well as solubility. In a study of five different buffers used for membrane proteins, for example, it was found that Tris buffer provided an optimal high-yielding lysis buffer for protein extraction for an integrin beta (2).
Beckwith also suggests using high throughput for buffer and detergent screening, if available, to reach desirable experimental conditions more quickly. He also notes that overall solubility of membrane proteins can be improved using purification tags to minimize aggregation and precipitation. “Maltose binding protein-tag and glutathione S-transferase-tag are often used for this purpose due to their high solubility, making it possible to purify the membrane protein using an affinity chromatography approach to yield high levels of purity after one step.”
Antibodies, nanobodies and ligands can also be used to stabilize membrane proteins (1). More recent technological developments are also helping scientists achieve greater success with membrane protein extraction, side-stepping traditional protocols with newer innovations. “Lipid nanodiscs provide a lipid bilayer into which membrane proteins can be reconstituted, and this has made it possible to perform research on membrane proteins using detergent-free buffers,” comments Beckwith.
Ensuring effective purification
Detergents, which are used to extract and purify membrane proteins by dissolving the biological membranes on which they sit, form soluble “proteomicelles” that can protect hydrophobic surfaces from water. Choice of detergent can significantly influence results and determining the most effective one for a particular application and membrane protein is essential (6).
A dependence on “trial and error,” as well as a potentially misleading dependency on ‘critical aggregation concentration’ to determine detergent concentration, means that selection of an appropriate detergent that facilitates purification can be tricky. Newer detergents such as oligoglycerol detergents have been proposed as a solution to these issues, being able to self-assemble in aqueous solution. These have the advantage of being able to be structurally “designed” and optimized in advance to effectively purify specific proteins (6).
“Use of analytical-grade chromatography tools makes it possible to maintain high sample concentrations for experimental use even with small volumes, which can be useful for membrane protein purification,” states Beckwith. “In addition, analyzing purified samples using surface plasmon resonance makes it possible to rapidly determine protein activity in a range of buffer/detergent conditions,” he concludes, meaning that scientists can accurately and quickly gauge success for the outcome of their membrane protein expression, extraction, and purification.
- Errasti-Murugarren,E., Bartoccioni, P., Palacín, M, 2021. Membrane Protein Stabilization Strategies for Structural and Functional Studies. Membranes 11:155.
- Muinao, T., et al., 2018. Cytosolic and Transmembrane Protein Extraction Methods of Breast and Ovarian Cancer Cells: A Comparative Study. J Biomol Techniques 21:71-8
- Mathieu, K., et al., 2019. Functionality of membrane proteins overexpressed and purified from E. coli is highly dependent upon the strain. Scientific Reports 9:2654.
- Swietnicki, W., 2006. Folding aggregated proteins into functionally active forms. Current Opinion Biotech. 17, 367–72.
- Bannwarth, M. and Schulz, G. E., 2003. The expression of outer membrane proteins for crystallization. Biochim. Biophys. Acta 1610, 37–45.
- Urner, L.H., et al., 2020. Modular detergents tailor the purification and structural analysis of membrane proteins including G-protein coupled receptors. Nat Commun 11:564.