Boggavarapu Rajendra, Hirschi Stephan, Harder Daniel, Meury Marcel, Ucurum Zöhre, Bergeron Marc J, Fotiadis Dimitrios
Institute of Biochemistry and Molecular Medicine, and Swiss National Centre of Competence in Research (NCCR) TransCure, University of Bern, Bühlstrasse 28, 3012, Bern, Switzerland.
Division of Cellular and Molecular Neuroscience, Institut Universitaire en Santé Mentale de Québec, Québec, QC, Canada, G1J 2G3.
Methods Mol Biol. 2016;1432:223-42. doi: 10.1007/978-1-4939-3637-3_14.
This protocol describes the isolation of recombinant human and mammalian membrane proteins expressed in Xenopus laevis frog oocytes for structural studies. The cDNA-derived cRNA of the desired genes is injected into several hundreds of oocytes, which are incubated for several days to allow protein expression. Recombinant proteins are then purified via affinity chromatography. The novelty of this method comes from the design of a plasmid that produces multi-tagged proteins and, most importantly, the development of a protocol for efficiently discarding lipids, phospholipids, and lipoproteins from the oocyte egg yolk, which represent the major contaminants in protein purifications. Thus, the high protein purity and good yield obtained from this method allows protein structure determination by transmission electron microscopy of single detergent-solubilized protein particles and of 2D crystals of membrane protein embedded in lipid bilayers. Additionally, a radiotracer assay for functional analysis of the expressed target proteins in oocytes is described. Overall, this method is a valuable option for structural studies of mammalian and particularly human proteins, for which other expression systems often fail.
本方案描述了从非洲爪蟾卵母细胞中表达的重组人源和哺乳动物膜蛋白的分离方法，用于结构研究。将所需基因的cDNA衍生的cRNA注射到数百个卵母细胞中，孵育数天以使蛋白质表达。然后通过亲和层析纯化重组蛋白。该方法的新颖之处在于设计了一种产生多标签蛋白的质粒，最重要的是，开发了一种从卵母细胞卵黄中有效去除脂质、磷脂和脂蛋白的方案，这些物质是蛋白质纯化中的主要污染物。因此，通过这种方法获得的高蛋白纯度和良好产量，使得能够通过对单一去污剂溶解的蛋白质颗粒以及嵌入脂质双层中的膜蛋白二维晶体进行透射电子显微镜观察来确定蛋白质结构。此外，还描述了一种用于对卵母细胞中表达的目标蛋白进行功能分析的放射性示踪测定法。总体而言，对于哺乳动物尤其是人类蛋白质的结构研究，该方法是一种有价值的选择，而其他表达系统在这方面常常失败。
