Improved expression and purification of human multidrug resistance protein MDR1 from baculovirus-infected insect cells
Improved expression and purification of human multidrug resistance protein MDR1 from baculovirus-infected insect cells
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DOI：
10.1016/j.pep.2009.02.010
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发表时间：
2009-07-01
影响因子：
1.6
通讯作者：
Kato, Hiroaki
中科院分区：
文献类型：
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作者：
Kodan, Atsushi;Shibata, Hiroyuki;Kato, Hiroaki
多药耐药蛋白MDR 1（P-glycoprotein/ABCB 1）是一种ATP依赖性的细胞毒性药物外排泵，与肿瘤对化疗药物的耐药性有关。为了实现其机制的三维结构分析，大量的高质量和同质的MDR 1蛋白是必不可少的。在这里，我们报告了一种具有成本效益的方法，用于大规模表达人MDR 1的杆状病毒/昆虫expressSF+细胞系统和替代的纯化方法，以保持MDR 1在单分散状态。经过广泛优化的洗涤剂，pH值，和添加剂，高产量（2.8 mg/L）的纯化的MDR 1获得固定化金属螯合亲和和尺寸排阻色谱与49%的回收率。纯化的MDR 1表现出特定的ATP水解酶活性（1.7 μ mol/min/mg）的底物，维拉帕米的存在下。该值比没有药物的基础活性高14倍。纯化的MDR 1的尺寸排阻色谱分析显示出单分散的洗脱曲线。该纯化方法为人MDR 1的结构和功能研究提供了合适的材料。(C)2009 Elsevier Inc. All rights reserved.
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.