Optimized purification of a heterodimeric ABC transporter in a highly stable form amenable to 2-D crystallization.
Optimized purification of a heterodimeric ABC transporter in a highly stable form amenable to 2-D crystallization.
复制标题
DOI：
10.1371/journal.pone.0019677
复制
发表时间：
2011
期刊：
影响因子：
3.7
通讯作者：
Jault JM
中科院分区：
文献类型：
--
作者：
Galián C;Manon F;Dezi M;Torres C;Ebel C;Lévy D;Jault JM
为了研究膜蛋白的结构和功能，需要优化的方法来实现膜蛋白的高产量表达、纯化和重组。我们先前报道了枯草芽孢杆菌的活性BmrC和BmrD蛋白在大肠杆菌中的高水平表达，以前命名为YheI和YheH。这些蛋白质是属于ABC（ATP结合盒）超家族的半转运蛋白，并且在体内结合以形成能够外排药物的功能性转运蛋白。在本报告中，高产量的纯化和功能重建的异源二聚体BmrC/BmrD实现。与其他更有效溶解转运蛋白的去污剂相比，十二烷基-β-D-麦芽糖苷（DDM）在通过亲和色谱纯化后将其保持在药物敏感和钒酸盐敏感的ATP酶活性状态。获得了大量的纯蛋白质，其通过分析超离心或凝胶过滤显示在溶液中形成单分散的异源二聚体，其在4 ° C下显著稳定超过一个月。使用不同脂质组合物的功能重建诱导ATP酶活性增加8倍（k cat ≥ 5 s-1）。我们进一步验证了纯化的BmrC/BmrD异二聚体的质量适合于结构分析，因为其在高蛋白密度下的重构导致2-D晶体的形成。负染色晶体的电子显微镜允许在20 μ m分辨率下计算投影图，揭示BmrC/BmrD可能在无菌环境中组装成寡聚体。
Optimized protocols for achieving high-yield expression, purification and reconstitution of membrane proteins are required to study their structure and function. We previously reported high-level expression in Escherichia coli of active BmrC and BmrD proteins from Bacillus subtilis, previously named YheI and YheH. These proteins are half-transporters which belong to the ABC (ATP-Binding Cassette) superfamily and associate in vivo to form a functional transporter able to efflux drugs. In this report, high-yield purification and functional reconstitution were achieved for the heterodimer BmrC/BmrD. In contrast to other detergents more efficient for solubilizing the transporter, dodecyl-ß-D-maltoside (DDM) maintained it in a drug-sensitive and vanadate-sensitive ATPase-competent state after purification by affinity chromatography. High amounts of pure proteins were obtained which were shown either by analytical ultracentrifugation or gel filtration to form a monodisperse heterodimer in solution, which was notably stable for more than one month at 4°C. Functional reconstitution using different lipid compositions induced an 8-fold increase of the ATPase activity (k cat∼5 s−1). We further validated that the quality of the purified BmrC/BmrD heterodimer is suitable for structural analyses, as its reconstitution at high protein densities led to the formation of 2-D crystals. Electron microscopy of negatively stained crystals allowed the calculation of a projection map at 20 Å resolution revealing that BmrC/BmrD might assemble into oligomers in a lipidic environment.