Detergent screening and purification of the human liver ABC transporters BSEP (ABCB11) and MDR3 (ABCB4) expressed in the yeast Pichia pastoris.
Detergent screening and purification of the human liver ABC transporters BSEP (ABCB11) and MDR3 (ABCB4) expressed in the yeast Pichia pastoris.
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DOI：
10.1371/journal.pone.0060620
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Schmitt L
中科院分区：
文献类型：
--
作者：
Ellinger P;Kluth M;Stindt J;Smits SH;Schmitt L
人肝ATP结合盒（ABC）转运体胆盐输出泵（BSEP/ABCB 11）和多药耐药蛋白3（MDR 3/ABCB 4）完成胆盐和磷脂酰胆碱跨肝细胞顶膜的转运。与ABCG 5/G8一致，这两种转运蛋白负责胆汁的形成，这些转运蛋白内的突变可导致严重的遗传性疾病。在这项研究中，我们报告了异源表达和纯化的人BSEP和MDR 3以及相应的C-末端GFP融合蛋白在酵母毕赤酵母中的表达。共聚焦激光扫描显微镜显示BSEP-GFP和MDR 3-GFP定位于巴斯德毕赤酵母的质膜上。此外，我们证明了人BSEP和MDR 3的首次纯化，分别产生每100 g湿细胞重量101 mg和106 mg。通过使用斑点印迹技术筛选超过100种洗涤剂，我们发现只有两性离子的脂质样洗涤剂如Fos-胆碱或Cyclofos能够以足够的量提取两种转运蛋白用于随后的功能分析。对于MDR 3，荧光检测尺寸排阻色谱（FSEC）筛选显示，增加Fos-胆碱的酰基链长度改善了单分散性。BSEP在正十二烷基-β-D-麦芽糖苷或Cymal-5中纯化后，用来自巴斯德毕赤酵母膜的Fos-胆碱16增溶，显示与ATP-琼脂糖结合。此外，洗涤剂溶解和纯化的MDR 3显示出底物诱导的ATP酶活性后，加入磷脂酰胆碱脂质。这些结果构成了进一步对人BSEP和MDR 3进行生化分析的基础，以阐明这些临床相关ABC转运蛋白的功能。
The human liver ATP-binding cassette (ABC) transporters bile salt export pump (BSEP/ABCB11) and the multidrug resistance protein 3 (MDR3/ABCB4) fulfill the translocation of bile salts and phosphatidylcholine across the apical membrane of hepatocytes. In concert with ABCG5/G8, these two transporters are responsible for the formation of bile and mutations within these transporters can lead to severe hereditary diseases. In this study, we report the heterologous overexpression and purification of human BSEP and MDR3 as well as the expression of the corresponding C-terminal GFP-fusion proteins in the yeast Pichia pastoris. Confocal laser scanning microscopy revealed that BSEP-GFP and MDR3-GFP are localized in the plasma membrane of P. pastoris. Furthermore, we demonstrate the first purification of human BSEP and MDR3 yielding ∼1 mg and ∼6 mg per 100 g of wet cell weight, respectively. By screening over 100 detergents using a dot blot technique, we found that only zwitterionic, lipid-like detergents such as Fos-cholines or Cyclofos were able to extract both transporters in sufficient amounts for subsequent functional analysis. For MDR3, fluorescence-detection size exclusion chromatography (FSEC) screens revealed that increasing the acyl chain length of Fos-Cholines improved monodispersity. BSEP purified in n-dodecyl-β-D-maltoside or Cymal-5 after solubilization with Fos-choline 16 from P. pastoris membranes showed binding to ATP-agarose. Furthermore, detergent-solubilized and purified MDR3 showed a substrate-inducible ATPase activity upon addition of phosphatidylcholine lipids. These results form the basis for further biochemical analysis of human BSEP and MDR3 to elucidate the function of these clinically relevant ABC transporters.
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影响因子：
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作者：
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通讯作者：
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