GFP-based optimization scheme for the overexpression and purification of eukaryotic membrane proteins in Saccharomyces cerevisiae
GFP-based optimization scheme for the overexpression and purification of eukaryotic membrane proteins in Saccharomyces cerevisiae
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DOI：
10.1038/nprot.2008.44
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发表时间：
2008-01-01
期刊：
影响因子：
14.8
通讯作者：
Iwata, So
中科院分区：
文献类型：
--
作者：
Drew, David;Newstead, Simon;Iwata, So
真核生物膜蛋白的大量生产往往是困难的，这是分析其生化和结构特征的主要障碍。迄今为止，酵母已经是最成功的异源过表达系统在生产真核细胞膜蛋白的高分辨率结构研究。为此，我们开发了一种快速筛选和纯化酿酒酵母真核细胞膜蛋白的方法。使用该方案，在1周内，可以通过同源重组将许多基因快速克隆到2 μ GFP融合载体中，并且使用全细胞和凝胶内荧光测定它们的过表达潜力。然后可以使用共聚焦显微镜和荧光尺寸排阻色谱法（FSEC）在几天内评估过量产生的真核细胞膜蛋白-GFP融合物的质量。该方案还详细说明了通过我们的质量标准的目标的纯化，并且可以在学术，结构基因组学或商业环境中扩大用于大量真核细胞膜蛋白。
It is often difficult to produce eukaryotic membrane proteins in large quantities, which is a major obstacle for analyzing their biochemical and structural features. To date, yeast has been the most successful heterologous overexpression system in producing eukaryotic membrane proteins for high-resolution structural studies. For this reason, we have developed a protocol for rapidly screening and purifying eukaryotic membrane proteins in the yeast Saccharomyces cerevisiae. Using this protocol, in 1 week many genes can be rapidly cloned by homologous recombination into a 2 mu GFP- fusion vector and their overexpression potential determined using whole-cell and in-gel fluorescence. The quality of the overproduced eukaryotic membrane protein-GFP fusions can then be evaluated over several days using confocal microscopy and fluorescence size-exclusion chromatography (FSEC). This protocol also details the purification of targets that pass our quality criteria, and can be scaled up for a large number of eukaryotic membrane proteins in either an academic, structural genomics or commercial environment.