High-Yield Preparation of Outer Membrane Protein Efflux Pumps by in Vitro Refolding is Concentration Dependent

Abstract

Overexpression of tripartite efflux pump systems in gram-negative bacteria is 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. A classical method for refolding outer membrane proteins involves unfolding inclusion bodies in urea followed by refolding in lipid or detergent micelles. However, that method has not yet been successful in refolding tripartite efflux pump TolC. Here, we find that refolding TolC from inclusion bodies requires an additional oligomerization enhancing step of sample concentration. We show that by our method of refolding, 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 TolC refolding is concentration dependent. We then extended our method to refolding 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 refolding protocols will promote correct refolding allowing for reliable, high-yield preparation of this family of proteins.

Keywords: Antibiotic resistance; In vitro folding; Outer membrane protein.

Fig. 1

How to refold TolC in vitro a The folded structure of outer membrane protein TolC from Escherichia coli (from PDB 1EK9 (Koronakis et al. 2000)). TolC is a homotrimer. Each color (red, green and blue) is a diferent chain. b TolC expression, purifcation, and refolding scheme.

Fig. 2

Refolding of TolC is concentration dependent. a Heat modifability assay to assess folding of TolC indicates that TolC transitions into trimers upon concentration. b TolC trimerization as a function of concentration. Representative SDS-PAGE showing the transition of the monomeric species to trimeric as concentration increases. Equilibrium constant calculated from four replicates and standard error reported.

Fig. 3

Refolded TolC binds to Col5. SEC chromatograms of TolC (purple), Col5 (cyan), and TolC+Col5 (black). TolC and Col5 were mixed at a 1:2 molar ratio 10 μM TolC:20 μM Col5. Peak fraction from 10.2 mL (red arrow) used for SDS-PAGE analysis. Sample was boiled as refected by only monomeric TolC species.

Fig. 4

Single particle cryoEM reveals properly refolded TolC. a Representative micrograph of nanodisc-embedded TolC with a scale bar in black representing 0.05 μm b Two-dimensional (2D) classes of nanodisc-embedded TolC in side and top views with white scale bar representing 0.01 μm c Ab initio reconstruction of nanodisc-embedded TolC (translucent gray), top and side view, with TolC crystal structure (PDB: 1EK9, (Koronakis et al. 2000)) rigidly docked into the density and colored by chain. Scale bar 1 nm.

Fig. 5

Refolding of CmeC is concentration dependent. a The folded structure of outer membrane protein CmeC from Campylobacter jejuni (from PDB 4MT4). CmeC is a homotrimer. Each color (pink, teal, and navy blue) is a different chain. b Heat modifability assay to assess folding of CmeC indicates that CmeC transitions into trimers upon concentration like TolC. c Size exclusion chromatography of CmeC (pink) versus TolC (black) d MALDI mass spectrometry of CmeC with (blue, top) and without (green bottom) covalent crosslinker disuccinimidyl suberate (DSS).

Fig. 6

Potential pathways of in vitro TolC refolding