The multidrug resistance half-transporter ABCG2 is purified as a tetramer upon selective extraction from membranes.
ABCG2 is a human membrane ATP-binding cassette half-transporter that hydrolyzes ATP to efflux a large number of chemotherapeutic agents. Several oligomeric states of ABCG2 from homodimers to dodecamers have been reported depending on the overexpression systems and/or the protocols used for purification. Here, we compared the oligomeric state of His(6)-ABCG2 expressed in Sf9 insect cells and in human Flp-In-293/ABCG2 cells after solubilization in mild detergents. His(6)-ABCG2 was purified through a new approach involving its specific recognition onto a functionalized lipid layer containing a Ni-NTA lipid. This approach allowed the purification of His-ABCG2 in presence of all solubilized membrane components that might be involved in the stabilisation of native oligomers and without requiring any additional washing or concentration passages. ABCG2 purified onto the NiNTA lipid surfaces were directly analyzed by electron microscopy and by biochemical assays. Altogether, our data are consistent with a tetrameric organization of ABCG2 when expressed in either heterologous Sf9 insect cells or in human homologous cells.
Dezi
Manuela
M
Institut Curie, Centre de Recherche, Paris, F-75231, France.
Fribourg
Pierre-Fréderic
PF
Di Cicco
Aurelie
A
Arnaud
Ophelie
O
Marco
Sergio
S
Falson
Pierre
P
Di Pietro
Attilio
A
Lévy
Daniel
D
D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000302:isolation & purification*; D000818:Animals; D002462:Cell Membrane / Q000737:chemistry*; D006801:Humans; D008854:Microscopy, Electron; D009363:Neoplasm Proteins / Q000737:chemistry / Q000302:isolation & purification*; D055503:Protein Multimerization; D018411:Spodoptera
C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D009363:Neoplasm Proteins