Expression of 25 Human ABC Transporters in the Yeast Pichia pastoris and Characterization of the Purified ABCC3 ATPase Activity
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- 作者：Maja Chloupková ; Amanda Pickert ; Jyh-Yeuan Lee ; Shiloe Souza ; Yenphuong T. Trinh ; Sara M. Connelly ; Mark E. Dumont ; Michael Dean ; Ina L. Urbatsch
- 刊名：Biochemistry
- 出版年：2007
- 出版时间：July 10, 2007
- 年：2007
- 卷：46
- 期：27
- 页码：7992 - 8003
- 全文大小：449K
- 年卷期：v.46,no.27(July 10, 2007)
- ISSN：1520-4995
文摘
Human ATP-binding cassette (ABC) transporters comprise a family of 48 membrane-spanningtransport proteins, many of which are associated with genetic diseases or multidrug resistance of cancers.In this study, we present a comprehensive approach for the cloning, expression, and purification of humanABC transporters in the yeast Pichia pastoris. We analyzed the expression of 25 proteins and demonstratethat 11 transporters, including ABCC3, ABCB6, ABCD1, ABCG1, ABCG4, ABCG5, ABCG8, ABCE1,ABCF1, ABCF2, and ABCF3, were expressed at high levels comparable to that of ABCB1 (P-glycoprotein).As an example of the purification strategy via tandem affinity chromatography, we purified ABCC3 (MRP3)whose role in the transport of anticancer drugs, bile acids, and glucuronides has been controversial. Theyield of ABCC3 was 3.5 mg/100 g of cells in six independent purifications. Purified ABCC3, activatedwith PC lipids, exhibited significant ATPase activity with a Vmax of 82 ± 32 nmol min-1 mg-1. TheATPase activity was stimulated by bile acids and glucuronide conjugates, reaching 170 ± 28 nmol min-1mg-1, but was not stimulated by a variety of anticancer drugs. The glucuronide conjugates ethinylestradiol-3-glucuronide and 17
-estradiol-17-glucuronide stimulated the ATPase with relatively high affinities(apparent Km values of 2 and 3 
M, respectively) in contrast to bile acids (apparent Km values of >130M), suggesting that glucuronides are the preferred substrates for this transporter. Overall, the availabilityof a purification system for the production of large quantities of active transporters presents a major stepnot only toward understanding the role of ABCC3 but also toward future structure-function analysis ofother human ABC transporters.
-estradiol-17-glucuronide stimulated the ATPase with relatively high affinities(apparent Km values of 2 and 3 M, respectively) in contrast to bile acids (apparent Km values of >130M), suggesting that glucuronides are the preferred substrates for this transporter. Overall, the availabilityof a purification system for the production of large quantities of active transporters presents a major stepnot only toward understanding the role of ABCC3 but also toward future structure-function analysis ofother human ABC transporters.