Institute of Biochemistry, Heinrich Heine University, Düsseldorf, Germany.
PLoS One. 2013 Apr 4;8(4):e60620. doi: 10.1371/journal.pone.0060620. Print 2013.
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.
人肝 ATP 结合盒 (ABC) 转运体胆汁盐输出泵 (BSEP/ABCB11) 和多药耐药蛋白 3 (MDR3/ABCB4) 完成胆汁盐和磷脂酰胆碱穿过肝细胞顶膜的转运。与 ABCG5/G8 一起，这两种转运体负责胆汁的形成，这些转运体的突变可导致严重的遗传性疾病。在这项研究中，我们报告了人 BSEP 和 MDR3 的异源过表达和纯化，以及相应的 C 端 GFP 融合蛋白在毕赤酵母中的表达。共焦激光扫描显微镜显示 BSEP-GFP 和 MDR3-GFP 定位于毕赤酵母的质膜上。此外，我们首次纯化了人 BSEP 和 MDR3，分别从 100 克湿细胞中获得约 1 毫克和 6 毫克。通过使用斑点印迹技术筛选了超过 100 种去污剂，我们发现只有两性离子、类脂去污剂，如 Fos-胆碱或 Cyclofos，才能以足够的量提取这两种转运体，用于后续的功能分析。对于 MDR3，荧光检测凝胶过滤色谱 (FSEC) 筛选显示，增加 Fos-胆碱的酰基链长度可提高均一性。从毕赤酵母膜中用 Fos-胆碱 16 溶解后，用正十二烷基-β-D-麦芽糖苷或 Cymal-5 纯化的 BSEP 显示与 ATP-琼脂糖结合。此外，添加磷脂酰胆碱脂质后，去污剂溶解和纯化的 MDR3 显示出底物诱导的 ATP 酶活性。这些结果为进一步的人 BSEP 和 MDR3 生化分析奠定了基础，以阐明这些具有临床意义的 ABC 转运体的功能。
