High yield preparation of outer-membrane protein efflux pumps by in vitro refolding is concentration dependent

Overexpression of tripartite efflux pump systems in gram-negative bacteria are a principal component of antibiotic resistance. High-yield purification of the outer membrane component of these systems will enable biochemical and structural interrogation of their mechanisms of action and allow testing of compounds that target them. However, preparation of these proteins is typically hampered by low yields requiring laborious large-scale efforts. If refolding conditions can be found, refolding these proteins from inclusion bodies can lead to increased yields as compared to membrane isolations. Here, we develop a concentration-dependent folding protocol for refolding TolC, the outer membrane component of the antibiotic efflux pump from Escherichia coli. We show that by our method of re-folding, homotrimeric TolC remains folded in SDS-PAGE, retains binding to an endogenous ligand, and recapitulates the known crystal structure by single particle cryoEM analysis. We find that a key factor in successful re-folding is a concentration dependence of TolC oligomerization. We extended the scheme to CmeC, a homologous protein from Campylobacter jejuni, and find that concentration-dependent oligomerization is a general feature of these systems. Because outer-membrane efflux pump components are ubiquitous across gram-negative species, we anticipate that incorporating a concentration step in re-folding protocols will promote correct refolding allowing for reliable, high-yield preparation of this family of proteins.