Clémençon Benjamin, Fine Michael, Schneider Philipp, Hediger Matthias A
Institute of Biochemistry and Molecular Medicine (IBMM), University of Bern, Bern, Switzerland; Swiss National Centre of Competence in Research (NCCR) TransCure, University of Bern, Bern, Switzerland.
Institute of Biochemistry and Molecular Medicine (IBMM), University of Bern, Bern, Switzerland; Swiss National Centre of Competence in Research (NCCR) TransCure, University of Bern, Bern, Switzerland.
Methods Enzymol. 2015;556:241-65. doi: 10.1016/bs.mie.2014.12.011. Epub 2015 Mar 20.
Progress toward elucidating the 3D structures of eukaryotic membrane proteins has been hampered by the lack of appropriate expression systems. Recent work using the Xenopus oocyte as a novel expression system for structural analysis demonstrates the capability of providing not only the significant amount of protein yields required for structural work but also the expression of eukaryotic membrane proteins in a more native and functional conformation. There is a long history using the oocyte expression system as an efficient tool for membrane transporter and channel expression in direct functional analysis, but improvements in robotic injection systems and protein yield optimization allow the rapid scalability of expressed proteins to be purified and characterized in physiologically relevant structural states. Traditional overexpression systems (yeast, bacteria, and insect cells) by comparison require chaotropic conditions over several steps for extraction, solubilization, and purification. By contrast, overexpressing within the oocyte system for subsequent negative-staining transmission electron microscopy studies provides a single system that can functionally assess and purify eukaryotic membrane proteins in fewer steps maintaining the physiological properties of the membrane protein.
缺乏合适的表达系统阻碍了在阐明真核膜蛋白三维结构方面取得进展。最近利用非洲爪蟾卵母细胞作为一种用于结构分析的新型表达系统开展的研究表明，它不仅能够提供结构研究所需的大量蛋白质产量，还能使真核膜蛋白以更天然和功能性的构象表达。将卵母细胞表达系统作为一种高效工具用于膜转运蛋白和通道的直接功能分析已有很长历史，但是机器人注射系统的改进和蛋白质产量优化使得所表达的蛋白质能够快速扩大规模，以便在生理相关的结构状态下进行纯化和表征。相比之下，传统的过表达系统（酵母、细菌和昆虫细胞）在提取、溶解和纯化过程中需要经过几个步骤的离液序列高盐剂条件。相比之下，在卵母细胞系统中进行过表达，随后用于负染色透射电子显微镜研究，提供了一个单一的系统，该系统能够以较少的步骤在维持膜蛋白生理特性的情况下对真核膜蛋白进行功能评估和纯化。
