Improved expression and purification of human multidrug resistance protein MDR1 from baculovirus-infected insect cells

Multidrug resistance protein MDR1 (P-glycoprotein/ABCB1) is an ATP-dependent efflux pump for various cytotoxic agents, and is implicated in the resistance of human tumors to chemotherapeutic drugs. To achieve the three-dimensional structural analysis for its mechanistic implications, large amounts o...

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Bibliographic Details
Published in:Protein expression and purification 2009-07, Vol.66 (1), p.7-14
Main Authors: Kodan, Atsushi, Shibata, Hiroyuki, Matsumoto, Takashi, Terakado, Kanako, Sakiyama, Keita, Matsuo, Michinori, Ueda, Kazumitsu, Kato, Hiroaki
Format: Article
Language:English
Subjects:
Animals
ATP-Binding Cassette, Sub-Family B, Member 1 - genetics
ATP-Binding Cassette, Sub-Family B, Member 1 - isolation & purification
ATP-Binding Cassette, Sub-Family B, Member 1 - metabolism
Baculoviridae - genetics
Baculoviridae - metabolism
Baculovirus
Buffers
Cell Line
Cell Membrane - chemistry
Enzyme Stability
Expression
Humans
Insect cell
Insecta
Membrane proteins
Purification
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Summary:Multidrug resistance protein MDR1 (P-glycoprotein/ABCB1) is an ATP-dependent efflux pump for various cytotoxic agents, and is implicated in the resistance of human tumors to chemotherapeutic drugs. To achieve the three-dimensional structural analysis for its mechanistic implications, large amounts of high-quality and homogeneous MDR1 protein are essential. Here we report a cost-effective method for large-scale expression of human MDR1 using a baculovirus/insect expressSF+ cell system and an alterative purification method to maintain MDR1 in a monodispersed state. After extensively optimizing the detergent, pH, and additives, a high yield (2.8 mg/L) of purified MDR1 was obtained by immobilized metal chelate affinity and size-exclusion chromatographies with 49% recovery. The purified MDR1 exhibited specific ATP hydrolase activity (1.7 μmol/min/mg) in the presence of a substrate, verapamil. This value was 14-fold greater than the basal activity without the drug. Size-exclusion chromatography analysis of purified MDR1 showed a monodispersed elution profile. The present purification method provides suitable material for structural and functional studies on human MDR1.
ISSN:1046-5928
1096-0279
DOI:10.1016/j.pep.2009.02.010