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 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 micromol/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.
Kodan
Atsushi
A
Membrane Dynamics Research Group, RIKEN Harima Institute at SPring-8, Sayo, Hyogo, Japan.
Shibata
Hiroyuki
H
Matsumoto
Takashi
T
Terakado
Kanako
K
Sakiyama
Keita
K
Matsuo
Michinori
M
Ueda
Kazumitsu
K
Kato
Hiroaki
H
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D000818:Animals; D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism*; D002021:Buffers; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry; D004795:Enzyme Stability; D006801:Humans; D007313:Insecta
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D002021:Buffers