2009年
Improved expression and purification of human multidrug resistance protein MDR1 from baculovirus-infected insect cells
Protein Expression and Purification
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- 巻
- 66
- 号
- 1
- 開始ページ
- 7
- 終了ページ
- 14
- 記述言語
- 英語
- 掲載種別
- 研究論文(学術雑誌)
- DOI
- 10.1016/j.pep.2009.02.010
- 出版者・発行元
- ACADEMIC PRESS INC ELSEVIER SCIENCE
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 mu mol/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. (C) 2009 Elsevier Inc. All rights reserved.
- リンク情報
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- DOI
- https://doi.org/10.1016/j.pep.2009.02.010
- J-GLOBAL
- https://jglobal.jst.go.jp/detail?JGLOBAL_ID=200902286965602030
- Web of Science
- https://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcAuth=JSTA_CEL&SrcApp=J_Gate_JST&DestLinkType=FullRecord&KeyUT=WOS:000265346000002&DestApp=WOS_CPL
- URL
- http://www.scopus.com/inward/record.url?eid=2-s2.0-63649083045&partnerID=MN8TOARS
- ID情報
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- DOI : 10.1016/j.pep.2009.02.010
- ISSN : 1046-5928
- J-Global ID : 200902286965602030
- ORCIDのPut Code : 58433922
- SCOPUS ID : 63649083045
- Web of Science ID : WOS:000265346000002