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Protein Expr Purif. 2019 Jul;159:60-68. doi: 10.1016/j.pep.2019.03.002. Epub 2019 Mar 6.

Large-scale purification of functional human P-glycoprotein (ABCB1).

Author information

1
Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892, USA.
2
Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892, USA. Electronic address: ambudkar@mail.nih.gov.

Abstract

Human P-glycoprotein (P-gp) is an ATP-binding cassette transporter that has been implicated in altering the pharmacokinetics of anticancer drugs in normal tissues and development of multidrug resistance in tumor cells via drug efflux. There is still no definitive explanation of the mechanism by which P-gp effluxes drugs. One of the challenges of large-scale purification of membrane transporters is the selection of a suitable detergent for its optimal extraction from cell membranes. In addition, further steps of purification can often lead to inactivation and aggregation, decreasing the yield of purified protein. Here we report the large-scale purification of human P-gp expressed in High-Five insect cells using recombinant baculovirus. The purification strategies we present yield homogeneous functionally active wild type P-gp and its E556Q/E1201Q mutant, which is defective in carrying out ATP hydrolysis. Three detergents (1,2-diheptanoyol-sn-glycero-3-phosphocholine, dodecyl maltoside and n-octyl-β-d-glucopyranoside) were used to solubilize and purify P-gp from insect cell membranes. P-gp purification was performed first using immobilized metal affinity chromatography, then followed by a second step of either anion exchange chromatography or size exclusion chromatography to yield protein in concentrations of 2-12 mg/mL. Size exclusion chromatography was the preferred method, as it allows separation of monomeric transporters from aggregates. We show that the purified protein, when reconstituted in proteoliposomes and nanodiscs, exhibits both basal and substrate or inhibitor-modulated ATPase activity. This report thus provides a convenient and robust method to obtain large amounts of active homogeneously purified human P-gp that is suitable for biochemical, biophysical and structural characterization.

KEYWORDS:

ABC transporter; ABCB1; ATP hydrolysis; Multidrug resistance; Nanodiscs; P-glycoprotein; Reconstitution

PMID:
30851394
PMCID:
PMC6600825
[Available on 2020-07-01]
DOI:
10.1016/j.pep.2019.03.002

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