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  • br Materials and methods br Results br Discussion


    Materials and methods
    Discussion Although large-scale purification of homogeneous and functional membrane proteins is challenging, it is essential for biochemical, biophysical and structural characterization. This is particularly true for human P-gp, as its atomic structure has only been recently solved [14,38]. We present here a highly reproducible large-scale purification method to generate large quantities of human WT P-gp protein and its double EQ mutant from High-Five insect cells. P-gp from humans, mice, and C. elegans had been purified by earlier methods with yields ranging from 0.006 mg to 0.7 mg protein [[39], [40], [41], [42], [43]]. Reports of large-scale purification of human P-gp are still rare. In one report, mouse P-gp (product of the mdr1a gene) was purified using Pichia pastoris as an expression system, yielding approximately 6 mg of protein at 1–2 mg/L concentration from 564 mg of total protein, using DDM [44]. Others have obtained enough protein for biophysical [45] and crystallization studies even though purification yields were not reported [43,[46], [47], [48]]. Recombinant baculovirus has been shown to provide a high expression of P-gp in Sf9 or High-Five insect Formoterol Hemifumarate [8,23]. Insect cells yield unglycosylated P-gp. Although the lipid composition of insect cells differs from the mammalian cell composition [49], the function of human P-gp is very well preserved [23,24,28,30]. Incorporation of tags at the C-terminus of P-gp has proven to be useful for the purification, including hexahistidine tags and GFP [14]. We chose to use the hexahistidine tag because it provides a rapid and easy purification through IMAC. When proteins are subjected to IMAC, metal ions are immobilized by chelation to an insoluble matrix, and then amino acids, particularly histidine, bind to the chelated ions. Changes in pH or addition of a competitive inhibitor allows elution of the bound proteins. Our results showed that resins containing either cobalt or nickel can be used for purification of P-gp, with comparable results. Others have obtained mouse P-gp suitable for high-resolution structures using different purification strategies. For example, Pichia pastoris membranes containing mouse ABCB1 with a hexahistidine tag at the C-terminus were solubilized using 4.5% Triton® X-100, and an Ni-NTA step using 0.04% sodium cholate and 0.0675% β-DDM for elution, followed by size exclusion chromatography [46]. Amphiphiles or β-sheet peptide assemblies in complex with lipids have also been used [58] to obtain structural information by electron microscopy. Previously, Pollock el al. [59] showed that addition of CHS improves the thermal stability of P-gp and reduces the fraction that corresponds to aggregated forms, likely due to reduction of the fluidity of the micelle. In our experience, the yield of recovery was comparable to that achieved without CHS. However, we found that protein purified in DDM/CHS had a higher basal ATP hydrolysis rate when compared to protein purified in DDM alone. This is most likely due to increased stability in the presence of mixed micelles of DDM and CHS. Even though detergent micelles can provide a good environment for P-gp structure characterization [14,46,48,55] it has been shown that they may affect the stability of P-gp and its ability to bind modulators in the transmembrane region [28]. For these reasons, incorporation of P-gp in proteoliposomes or nanodiscs is preferable to study its properties in a membrane environment [21]. Membrane proteins can be reconstituted into lipid bilayers by detergent removal. P-gp has been reconstituted in both proteoliposomes and giant liposomes [30,52]. Here we demonstrate that purified P-gp can be easily reconstituted in proteoliposomes and nanodiscs in a functional form.
    Declarations of interest
    Author contributions
    Introduction Hepatitis C virus (HCV), a member of the Flaviviridae family, is one of the leading causes of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma worldwide. HCV, an enveloped virus with an approximately 9.6 kb long, positive stranded RNA genome, encodes a single polyprotein of about 3010 amino acids. Post-translationally, this polyprotein is cleaved (Fields et al., 2013) to generate at least 11 structural and non-structural proteins. Structural proteins include two viral glycoproteins, E1 and E2 (Yagnik et al., 2000) located at the N terminus of the polyprotein (Moradpour and Penin, 2013). HCV heterodimer E1/E2, the complex responsible for viral entry and tropism, are type I transmembrane proteins with an N terminal ectodomain and C- terminal transmembrane domain (TMD). The exact role of E1 in viral entry is poorly understood, but is believed to be related to the fusion process (Boo et al., 2012; Callens et al., 2005). E2, however, is currently the better characterized subunit where it plays pivotal roles in HCV entry, i.e. interaction with entry factors like scavenger receptor BI (SR-B1) (Scarselli et al., 2002), tetraspanin CD81 (Pileri et al., 1998) along with several tight junction proteins, Claudin-1 (Evans et al., 2007) and occludin receptors (Ciesek et al., 2011). E2 is also one of the most diverse HCV proteins characterized by two hypervariable regions (HVR), HVR1 and HVR2. Previous reports have shown that both HVR1 and HVR2 are important for the interaction with the cellular receptor protein CD81 (Callens et al., 2005; McCaffrey et al., 2011; Roccasecca et al., 2003), a 26 kDa integral membrane protein (Pileri et al., 1998). The receptor is essential for HCV glycoprotein mediated entry for all genotypes (Roccasecca et al., 2003). The CD81 binding site in E2 has been investigated in many studies primarily using genotype 1a (strain H77) and 2a (using JFH-1) (Callens et al., 2005; Roccasecca et al., 2003).