Functional high level expression of cytochrome P450 CYP2D6 using baculoviral expression systems
Functional high level expression of cytochrome P450 CYP2D6 using baculoviral expression systems
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DOI：
10.1006/abbi.1996.0154
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发表时间：
1996-04-01
影响因子：
3.9
通讯作者：
Wolf, CR
中科院分区：
文献类型：
--
作者：
Paine, MJI;Gilham, D;Wolf, CR
细胞色素P-450 CYP 2D 6在许多广泛使用的治疗药物（包括β-肾上腺素能拮抗剂、抗抑郁药和三环类抗抑郁药）的代谢中起核心作用。已经构建了含有全长人CYP 2D 6 cDNA的重组杆状病毒，并用于在草地贪夜蛾（Spodoptera frugiperda，Sf 9）细胞中表达CYP 2D 6。使用多角体蛋白或碱性蛋白启动子已经产生了高水平的重组蛋白（0.05-0.20 nmol/mg细胞蛋白; 0.05-0.15 nmol/升）。该酶对CYP 2D 6底物（如丁呋洛尔和美托洛尔）具有催化活性。为了优化催化活性，将人还原酶与CYP 2D 6在Sf 9细胞中共表达;还原酶活性在1000-1500单位/mg细胞蛋白的范围内，而光谱活性CY 2D 6在10-20 pmol/mg细胞蛋白的范围内。丁呋洛尔代谢的Km和K-cat值分别估计为4.7 μ M和12.23 min（-1）。使用常规的极晚期启动子如多角体蛋白启动子产生大比例的失活CYP 2D 6。使用在杆状病毒感染周期早期有活性的“晚期"基本启动子在一定程度上避免了这个问题。功能性CYP 2D 6的产量至少与非常晚的启动子一样高，但无活性蛋白质的比例减少，丁呋洛尔羟化酶活性可以直接通过HPLC分析补充丁呋洛尔的细胞培养基来测量，我们已经开发了一个平板测定系统，该系统提供了一种简单的方法，用于分析使用Sf 9细胞的药物代谢反应。使用杆状病毒的表达提供了一个有价值的来源的功能性CYP 2D 6的工作，旨在阐明酶的结构和功能。(C)出版社：Academic Press，Inc.
Cytochrome P-450 CYP2D6 plays a central role in the metabolism of many widely used therapeutic drugs including beta-adrenergic antagonists, antiarrhythmics, and tricyclic antidepressants. Recombinant baculoviruses have been constructed containing the full-length human CYP2D6 cDNA and used to express CYP2D6 in Spodoptera frugiperda (Sf9) cells. High levels of recombinant protein have been produced using either polyhedrin or basic protein promoters (0.05-0.20 nmol/mg cell protein; 0.05-0.15 nmol/liter). The enzyme is catalytically active toward CYP2D6 substrates such as bufuralol and metoprolol. In order to optimize catalytic activity human reductase was coexpressed with CYP2D6 in Sf9 cells; reductase activity was in the region of 1000-1500 units per mg cell protein, while spectrally active CY2D6 was in the range 10-20 pmol/mg cell protein. The K-m and K-cat values for bufuralol metabolism were estimated as 4.7 mu M and 12.23 min(-1), respectively. The use of the conventional very late promoters such as the polyhedrin promoter generate a large proportion of inactive CYP2D6. The problem was to a degree circumvented using the ''late'' basic promoter which is active earlier in the baculovirus infection cycle. The yield of functional CYP2D6 was at least as high as with very late promoters, but the proportion of inactive protein was reduced, Bufuralol hydroxylase activity could be measured directly by HPLC analysis of cell culture media supplemented with bufuralol, and we have developed a plate assay system which provides a simple method for the analysis of drug metabolism reactions using Sf9 cells. Expression using baculovirus provides a valuable source of functional CYP2D6 for work aimed at elucidating the structure and function of the enzyme. (C) 1996 Academic Press, Inc.