Unravelling the refolding of membrane proteins, one of the most challenging therapeutic targets

Abstract

Being involved in many essential cellular processes, Membrane Proteins (MPs) constitute one of the most important therapeutic targets, such as G protein-coupled receptors (GPCR), small molecule transporter or ion channels [1-3]. A detailed knowledge of their structure and activity is essential to design new therapeutic or biomimetic agents and therefore plays a significant role in the drug discovery process.
The major difficulties frequently encountered during MPs in vitro study are mainly due to their partially hydrophobic surfaces, flexibility and lack of stability. As a consequence, less than 1% of all solved protein crystal structures are MPs [4], witnessing how challenging is their expression, solubilisation, purification and crystallisation. Protein overexpression in E. coli is a very common method as it is quite inexpensive, easy to use, and can provide results in a reasonable lapse of time. However, the production of MPs from bacterial membrane is often toxic, and generally expression yields tend to be limited.
A well-established alternative is based on the formation of inactive aggregates called inclusion bodies (IBs) in the cell cytosol. Subsequently, a refolding step from IB is needed but it often turns to be a difficult task [5]. Whilst the refolding of soluble globular proteins from IB has become routine, much fewer examples of MPs in vitro refolding have indeed been reported.