Assessing protein-lipid interactions with a low-cost, accessible centrifugation assay.

Abstract

Assessing protein insertion and association with membranes is often a critical step that follows protein synthesis for both fundamental studies on protein folding and structure as well as translational applications that harness proteins for their activity. Traditionally, membrane protein association with membranes involves ultracentrifugation, which can be time-consuming and inaccessible in low-resource scientific environments. In this study, we develop an accessible method to purify vesicle-integrated cell-free expressed proteins from unincorporated protein or lysed membranes. We use a table-top microcentrifuge, capable of reaching speeds up to 21,130 × g, and a sucrose gradient to effectively separate the bulk of the cell-free expression components from proteoliposomes. We validate our approach can be used to measure membrane association of a variety of proteins, such as peripheral and transmembrane proteins as well as lipid-specific proteins, and that our method can be extended to membrane proteins derived from cellular membranes. Our approach provides a more accessible, cost-effective, and low-volume alternative for isolating proteoliposomes from misfolded and unassociated membrane proteins that should be applicable for fundamental biophysical studies and applications involving cell-free expressed membrane proteins.