A major hurdle in purifying a membrane protein is

A major hurdle in purifying a membrane protein is finding a detergent that can preserve the protein, after releasing it from a given membrane, in a stable and non-aggregated state during purification steps [18,50]. The ability of various detergents used to solubilize and purify membrane proteins has been reviewed [42], and in particular, the efficacy of different detergents to solubilize P-gp has been compared in previous publications [10,29,[51], [52], [53]]. Generally during the solubilization step, the detergent is used at >20 ✕ CMC to ensure spontaneous micelle formation. However, during purification, the concentration of detergent employed is usually just above the CMC. High concentrations of detergent can affect the integrity of the protein. Efforts to develop detergent-free isolation of membrane proteins are still under development [54]. Unfortunately, there is no single universal detergent that will work for all membrane proteins. For example, P-gps from murine lymphoid leukemia P388/ADR25, rat hepatoma AS30-D/COL10 and human lymphoblastic leukemia CEM/VLB5 are highly adaptable to extreme denaturing conditions in which solubilization and purification are carried out in the presence of SDS [19]. However, their function was significantly diminished when the protein was reconstituted in lipid vesicles. Lauryl maltose neopetyl glycol/CHS (LMNG/CHS) has recently been used for purification of a mouse/human P-gp chimera and for resolution of its structure by cryo-EM [47]. However, we found that with this detergent, the yield of solubilized human P-gp from insect cell membranes was considerably lower compared to the yield with DDM (data not shown). As noted in our earlier reports [23,30], DHPC, DDM and OG in combination with added 1 646 were each found to be effective detergents with reference to their ability to solubilize P-gp. They can be used interchangeably for column chromatography and can be removed during reconstitution in proteoliposomes or nanodiscs. These detergents also preserve the ATPase activity of P-gp. Here, we show that for preparation of homogeneous and biologically functional proteins, stability can be achieved by solubilizing the insect-cell membranes in either DHPC, DDM or OG. However, the choice of detergent in the second step of purification is critical for applications such as crystallization and functional assessment. Generally, detergents with lower CMCs are preferred for structural studies. Particularly, DDM has been widely proven to be a good detergent for P-gp structural characterization [14,46,48,55]. For these reasons, DDM was our detergent of choice for the second purification step. This detergent belongs to the class of alkyl maltosides and contains a hydrophilic maltose headgroup and a hydrophobic alkyl chain. DDM has a CMC of 0.17 mM (0.0087%), which is several orders of magnitude lower than OG or DHPC [56]. Apparently, the inhibition of ATPase activity of P-gp purified from Chinese hamster ovary (CHRB30 cells) by DDM detergent above CMC level could be prevented when used in combination with phospholipid mixtures. Even though the CMC and aggregation number (i.e., the number of detergent molecules per micelle) are specific to each detergent in a given buffer condition [57], the DDM present in the concentrated final column fraction containing human P-gp is much higher than CMC, which helps to prevent the aggregation of homogeneous preparations of human P-gp at higher concentrations. We have previously shown that the ATP hydrolysis behavior of P-gp is affected by DDM micelles [28]. The yield of P-gp or its mutant EQ protein and specific activity of ATPase activity were dependent on the type of detergent used in the final step. Though most non-ionic detergents readily solubilize P-gp, they significantly inhibit ATPase activity [39]. In addition to DDM or DHPC detergents, we found that the presence of 150 mM NaCl and 20% glycerol [36], an osmolyte, increases the stability during solubilization and throughout the process of purification and storage at −80 °C.