Chloupková Maja, Pickert Amanda, Lee Jyh-Yeuan, Souza Shiloe, Trinh Yenphuong T, Connelly Sara M, Dumont Mark E, Dean Michael, Urbatsch Ina L
Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas 79430-6540, USA.
Biochemistry. 2007 Jul 10;46(27):7992-8003. doi: 10.1021/bi700020m. Epub 2007 Jun 15.
Human ATP-binding cassette (ABC) transporters comprise a family of 48 membrane-spanning transport 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 human ABC transporters in the yeast Pichia pastoris. We analyzed the expression of 25 proteins and demonstrate that 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. The yield of ABCC3 was 3.5 mg/100 g of cells in six independent purifications. Purified ABCC3, activated with PC lipids, exhibited significant ATPase activity with a Vmax of 82 +/- 32 nmol min-1 mg-1. The ATPase activity was stimulated by bile acids and glucuronide conjugates, reaching 170 +/- 28 nmol min-1 mg-1, but was not stimulated by a variety of anticancer drugs. The glucuronide conjugates ethinylestradiol-3-glucuronide and 17beta-estradiol-17-glucuronide stimulated the ATPase with relatively high affinities (apparent Km values of 2 and 3 microM, respectively) in contrast to bile acids (apparent Km values of >130 microM), suggesting that glucuronides are the preferred substrates for this transporter. Overall, the availability of a purification system for the production of large quantities of active transporters presents a major step not only toward understanding the role of ABCC3 but also toward future structure-function analysis of other human ABC transporters.
人类ATP结合盒（ABC）转运蛋白家族由48种跨膜转运蛋白组成，其中许多与遗传疾病或癌症的多药耐药性有关。在本研究中，我们提出了一种在酵母毕赤酵母中克隆、表达和纯化人类ABC转运蛋白的综合方法。我们分析了25种蛋白的表达情况，证明包括ABCC3、ABCB6、ABCD1、ABCG1、ABCG4、ABCG5、ABCG8、ABCE1、ABCF1、ABCF2和ABCF3在内的11种转运蛋白的表达水平与ABCB1（P-糖蛋白）相当。作为通过串联亲和层析进行纯化策略的一个例子，我们纯化了ABCC3（多药耐药相关蛋白3），其在抗癌药物、胆汁酸和葡糖醛酸苷转运中的作用一直存在争议。在六次独立纯化中，ABCC3的产量为3.5 mg/100 g细胞。用磷脂酰胆碱（PC）脂质激活的纯化ABCC3表现出显著的ATP酶活性，Vmax为82±32 nmol min-1 mg-1。胆汁酸和葡糖醛酸苷结合物可刺激ATP酶活性，达到170±28 nmol min-1 mg-1，但多种抗癌药物不能刺激该活性。与胆汁酸（表观Km值>130 μM）相比，葡糖醛酸苷结合物乙炔雌二醇-3-葡糖醛酸苷和17β-雌二醇-17-葡糖醛酸苷以相对较高的亲和力（表观Km值分别为2和3 μM）刺激ATP酶，表明葡糖醛酸苷是该转运蛋白的首选底物。总体而言，用于大量生产活性转运蛋白的纯化系统的可用性不仅是理解ABCC3作用的重要一步，也是未来对其他人类ABC转运蛋白进行结构-功能分析的重要一步。
