Department of Chemistry, The Pennsylvania State University, University Park, PA, United States.
Department of Chemistry, The Pennsylvania State University, University Park, PA, United States.
Methods Enzymol. 2021;650:119-157. doi: 10.1016/bs.mie.2021.02.004. Epub 2021 Mar 19.
Recent work has revealed that certain lanthanides-in particular, the more earth-abundant, lighter lanthanides-play essential roles in pyrroloquinoline quinone (PQQ) dependent alcohol dehydrogenases from methylotrophic and non-methylotrophic bacteria. More recently, efforts of several laboratories have begun to identify the molecular players (the lanthanome) involved in selective uptake, recognition, and utilization of lanthanides within the cell. In this chapter, we present protocols for the heterologous expression in Escherichia coli, purification, and characterization of many of the currently known proteins that comprise the lanthanome of the model facultative methylotroph, Methylorubrum extorquens AM1. In addition to the methanol dehydrogenase XoxF, these proteins include the associated c-type cytochrome, XoxG, and solute binding protein, XoxJ. We also present new, streamlined protocols for purification of the highly selective lanthanide-binding protein, lanmodulin, and a solute binding protein for PQQ, PqqT. Finally, we discuss simple, spectroscopic methods for determining lanthanide- and PQQ-binding stoichiometry of proteins. We envision that these protocols will be useful to investigators identifying and characterizing novel members of the lanthanome in many organisms.
最近的研究揭示，某些镧系元素 - 特别是更丰富的轻镧系元素 - 在依赖吡咯喹啉醌（PQQ）的甲醇脱氢酶中发挥着重要作用，这些酶来自甲基营养型和非甲基营养型细菌。最近，几个实验室的努力已经开始识别参与选择性摄取、识别和利用细胞内镧系元素的分子参与者（镧系元素组）。在本章中，我们提供了在大肠杆菌中异源表达、纯化和表征许多目前已知的蛋白质的方案，这些蛋白质构成了模式兼性甲基营养菌 Methylorubrum extorquens AM1 的镧系元素组。除了甲醇脱氢酶 XoxF 外，这些蛋白质还包括相关的 c 型细胞色素 XoxG 和溶质结合蛋白 XoxJ。我们还提出了新的简化方案，用于纯化高度选择性的镧系元素结合蛋白 lanmodulin 和 PQQ 的溶质结合蛋白 PqqT。最后，我们讨论了用于确定蛋白质与镧系元素和 PQQ 结合化学计量的简单光谱方法。我们设想，这些方案将对鉴定和表征许多生物体中镧系元素组的新成员的研究人员有用。
