Detergent-free purification of ABC (ATP-binding-cassette) transporters

Abstract

Abstract:ABC (ATP Binding Cassette) transporters carry out many vital functions and are involved in numerous diseases, but study of the structure and function of these proteins is often hampered by their large size and membrane location. Membrane protein purification usually utilises detergents to solubilise the protein from the membrane, effectively removing it from its native lipid environment. Subsequently lipids have to be added back and detergent removed to reconstitute the protein into a lipid bilayer. We present here the application of a new methodology for the extraction and purification of ABC transporters without the use of detergent, instead using a styrene maleic acid co-polymer (SMA). SMA inserts in a bilayer and assembles into discrete particles, essentially solubilising the membrane into small discs of bilayer encircled by polymer, termed SMA lipid particles (SMALPs). We show that this polymer can extract several eukaryotic ABC transporters; P-glycoprotein (ABCB1), MRP1 (ABCC1), MRP4 (ABCC4), ABCG2 and CFTR (ABCC7), from a range of different expression systems. The SMALP encapsulated ABC transporters can be purified by affinity chromatography, and are able to bind ligands comparably to those in native membranes or detergent micelles. A greater degree of purity and enhanced stability is seen compared to detergent solubilisation. This study demonstrates that eukaryotic ABC transporters can be extracted and purified without ever being removed from their lipid bilayer environment, opening up a wide range of possibilities for the future study of their structure and function. Summary statement:A styrene maleic acid copolymer can be effectively used to extract and purify large eukaryotic transmembrane proteins in the absence of detergents, forming small bilayer discs encapsulating the protein, which have great potential for future structure & function studies.

Overcoming bottlenecks in the membrane protein structural biology pipeline

“…Proteins within SMALPs have been shown to bind ligands comparably to those in the native membrane [44]. The use of SMALPs has facilitated improved thermostability and homogeneity of membrane proteins such as P-glycoprotein (P-gp) and the adenosine A2a receptor [39,42,44]. To date no crystal structure has been reported using SMALPs but both negative stain and cryo-EM low resolution structural maps have been determined [39,43].…”

“…During the formation of SMALPs from a biological membrane, membrane proteins can become trapped inside these discs (as shown in Figure 1D), solubilising them into small particles (much like nanodiscs when using membrane scaffolding proteins), but without the need for detergent at any stage. The proteins within a SMALP can be effectively purfied using affinity chromatography [39,[42][43][44]. The particles are stable once formed so there is no need to supplement buffers during purification as there is when using detergents; this is a major cost advantage.…”

“…The use of SMALPs has facilitated improved thermostability and homogeneity of membrane proteins such as P-glycoprotein (P-gp) and the adenosine A2a receptor [39,42,44]. To date no crystal structure has been reported using SMALPs but both negative stain and cryo-EM low resolution structural maps have been determined [39,43]. Unlike MSP-nanodiscs, the polymer does not show significant extra-electron density; EM shows a very small annulus of lipids and polymer.…”

“…This copolymer has the ability to solubilise lipid membranes into nanodisc-like structures termed SMA lipid particles (SMALPs; also known as native nanodiscs or lipodisqs), where a small disc of lipid bilayer is encircled by the SMA co-polymer [39][40][41]. During the formation of SMALPs from a biological membrane, membrane proteins can become trapped inside these discs (as shown in Figure 1D), solubilising them into small particles (much like nanodiscs when using membrane scaffolding proteins), but without the need for detergent at any stage.…”

Bacterial Reaction Centers Purified with Styrene Maleic Acid Copolymer Retain Native Membrane Functional Properties and Display Enhanced Stability

“…A recently developed alternative is the use of an amphipathic styrene maleic acid (SMA) copolymer (Figure 1 A) that is able to remove the protein from a membrane with its associated lipids intact in the form of a protein/lipid nanodisc bound by the polymer. [2][3][4][5] This approach has been used to successfully solubilize membrane proteins from artificial liposomes [2,6] and native membranes, [7,8] and is one of a number of alternatives being developed for housing integral membrane proteins outside the native membrane. [9] Figure 1.…”