Technical Guide: Conducting In Vitro Assays with Transmembrane Proteins

Transmembrane proteins account for approximately 50% of the total mass in a lipid bilayer and serve as front line communicators and transporters. Transmembrane proteins (TMPs) are integral membrane proteins permanently attached to a biological membrane. TMPs typically contain three major regions – an extracellular domain, membrane domain and an intracellular domain. Although TMPs are prevalent drug targets, they are inherently difficult to work with, especially when conducting in vitro assays. Level of purity, solubility and conformational stability are some major pain points when conducting in vitro assays with transmembrane proteins. Fortunately, there are solutions and techniques available to researchers wanting to conduct in vitro assays with transmembrane proteins. 

30% of all proteins in the cell are transmembrane proteins and are common therapeutic targets. Therefore, it is essential to employ a variety of techniques including animal, in vivo and in vitro assays to characterize all behaviours of TMPs. A well rounded technical portfolio including animal, in vivo and in vitro assays will accomplish two things:

1. Give you, as a researcher, much more insight into the unique behaviours of the protein(s) and drugs under study.

2. Give reviewers more confidence in your work and in turn, a higher probability of accepting your work.

With that said, using in vitro techniques when working with transmembrane proteins has historically been a difficult task. Fortunately, there are a number of solutions available to researchers.

Purity and Concentration

One barrier when attempting to work in vitro with transmembrane proteins is purity and concentrations. TMPs are often not expressed in cell membranes in high enough quantities to purify for in vitro assays (except for Rhodopsin). If this is true for you and your intended project, consider one of the following:

• Metal ion expression tags and metal affinity chromatography
• High-yield expression systems (i.e. HEK293F and HEK293S cell lines)
• Gene synthesis/transmembrane protein supplier

Solubility

Another limiting factor for conducting in vitro assays with transmembrane proteins is solubility. At higher concentrations, TMPs tend to aggregate. These are some suggestions and solutions to overcome solubility issues with transmembrane proteins:

• Adjust the pI and pH of the buffer conditions
• Start by adding a simple surfactant to the buffer such as SDS, Tween or TritonX100 (without denaturing the protein under scrutiny)
• If required, add a more complex surfactant to the buffer such as C12E8 and DHPC
• Express your protein(s) with an MBP tag

Native Conformation

If a protein does not retain its native conformation, the protein will lose it’s activity and functionality, becoming useless. This is a primary concern when working with transmembrane proteins. Historically, once a transmembrane protein has been removed from the lipid bilayer environment, conformation and thus, functionality will be lost. However, there are a few technical and commercially-available solutions to mitigate this from happening:

• Reconstitution of transmembrane proteins into liposomes
• Protein suppliers who guarantee conformational stability (yes, these companies exist!) 
• Reconstitution of transmembrane proteins into nanodiscs
• Truncating intracellular and  extracellular domains from the full-length protein and employing adequate positive and negative controls

OpenSPR™ Gets You The Data You Need To Publish

It should be re-noted that these suggestions and solutions are to maximize the efficacy and efficiency of your intended in vitro work with transmembrane proteins. Importantly, many in vitro techniques include binding assays, such as Surface Plasmon Resonance (SPR). SPR is a label-free technique that provides binding kinetics data in real-time. More and more reviewers are asking for quantitative binding kinetics data over simple yes/no binding data for biomolecular interactions. For example, these researchers used the OpenSPR™ to quantitatively assess the kinetics of a transmembrane receptor. Traditionally, binding kinetics data has been extremely inaccessible, but with the OpenSPR™, you can obtain the same quality data that reviewers want for a fraction of the cost.

Let us help you publish your next big discovery and bring your breakthrough to the market!

References

1. Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., & Walter, P. (2002). Molecular biology of the cell (4th ed.). New York: Garland Science. doi: https://www.ncbi.nlm.nih.gov/books/NBK21054/
   
2. Singh, S. K., & Dixit, T. (2014). Pharmacogenomics in Anesthesia. Handbook of Pharmacogenomics and Stratified Medicine, 815–833. doi: 10.1016/b978-0-12-386882-4.00035-9
   
3. Welch, M., Villalobos, A., Gustafsson, C., & Minshull, J. (2011). Designing Genes for Successful Protein Expression. Methods in Enzymology Synthetic Biology, Part B – Computer Aided Design and DNA Assembly, 498, 43–66. doi: 10.1016/b978-0-12-385120-8.00003-6