Université de Montpellier, IRIM, CNRS, Montpellier, France.
Université de Montpellier, IRIM, CNRS, Montpellier, France; Department of Molecular Biology and Genetics, University of Aarhus, 8000, Aarhus, Denmark.
Protein Expr Purif. 2022 Mar;191:106014. doi: 10.1016/j.pep.2021.106014. Epub 2021 Nov 9.
Mycobacteria possess a complex and waxy cell wall comprising a large panel of glycolipids. Among these, trehalose monomycolate (TMM) represents abundant and crucial components for the elaboration of the mycomembrane. TMM is synthesized in the cytoplasmic compartment and translocated across the inner membrane by the MmpL3 transporter. Inhibitors impeding TMM transport by targeting MmpL3 show great promises as new antimycobacterials. The recent X-ray or Cryo-EM structures of MmpL3 complexed to TMM or its inhibitors have shed light on the mechanisms of TMM transport and inhibition. So far, purification procedures mainly involved the use of n-Dodecyl-ß-d-Maltopyranoside to solubilize and stabilize MmpL3 from Mycobacterium smegmatis (MmpL3) or Lauryl Maltose Neopentyl Glycol for MmpL3 from Mycobacterium tuberculosis. Herein, we explored the possibility to solubilize and stabilize MmpL3 with other detergents. We demonstrate that several surfactants from the ionic, non-ionic and zwitterionic classes are prone to solubilize MmpL3 expressed in Escherichia coli. The capacity of these detergents to stabilize MmpL3 was evaluated by size-exclusion chromatography and thermal stability. This study unraveled three new detergents DM, LDAO and sodium cholate that favor solubilization and stabilization of MmpL3 in solution. In addition, we report a protocol that allows reconstitution of MmpL3 into peptidiscs.
分枝杆菌具有复杂的蜡质细胞壁，包含大量的糖脂。在这些糖脂中，海藻糖单脂 (TMM) 是构成完整的细胞膜的重要成分。TMM 在细胞质中合成，并通过 MmpL3 转运体穿过内膜转运。靶向 MmpL3 以阻碍 TMM 转运的抑制剂作为新型抗分枝杆菌药物具有很大的应用前景。最近，MmpL3 与 TMM 或其抑制剂复合物的 X 射线或冷冻电镜结构揭示了 TMM 转运和抑制的机制。到目前为止，纯化程序主要涉及使用正十二烷基-β-D-麦芽糖苷（n-Dodecyl-β-D-Maltopyranoside）从耻垢分枝杆菌（MmpL3）或月桂基麦芽糖新戊二醇（Lauryl Maltose Neopentyl Glycol）中溶解和稳定 MmpL3，用于从结核分枝杆菌中提取 MmpL3。在此，我们探讨了使用其他去污剂溶解和稳定 MmpL3 的可能性。我们证明，来自离子型、非离子型和两性离子型的几种表面活性剂易于溶解在大肠杆菌中表达的 MmpL3。通过凝胶过滤色谱法和热稳定性评估了这些去污剂稳定 MmpL3 的能力。本研究揭示了三种新的去污剂 DM、LDAO 和胆酸钠，它们有利于 MmpL3 在溶液中的溶解和稳定。此外，我们还报告了一种将 MmpL3 重新组装成肽盘的方案。
