Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90095, USA.
Protein Sci. 2010 Apr;19(4):868-80. doi: 10.1002/pro.365.
Low expression and instability during isolation are major obstacles preventing adequate structure-function characterization of membrane proteins (MPs). To increase the likelihood of generating large quantities of protein, C-terminally fused green fluorescent protein (GFP) is commonly used as a reporter for monitoring expression and evaluating purification. This technique has mainly been restricted to MPs with intracellular C-termini (C(in)) due to GFP's inability to fluoresce in the Escherichia coli periplasm. With the aid of Glycophorin A, a single transmembrane spanning protein, we developed a method to convert MPs with extracellular C-termini (C(out)) to C(in) ones providing a conduit for implementing GFP reporting. We tested this method on eleven MPs with predicted C(out) topology resulting in high level expression. For nine of the eleven MPs, a stable, monodisperse protein-detergent complex was identified using an extended fluorescence-detection size exclusion chromatography procedure that monitors protein stability over time, a critical parameter affecting the success of structure-function studies. Five MPs were successfully cleaved from the GFP tag by site-specific proteolysis and purified to homogeneity. To address the challenge of inefficient proteolysis, we explored expression and purification conditions in the absence of the fusion tag. Contrary to previous studies, optimal expression conditions established with the fusion were not directly transferable for overexpression in the absence of the GFP tag. These studies establish a broadly applicable method for GFP screening of MPs with C(out) topology, yielding sufficient protein suitable for structure-function studies and are superior to expression and purification in the absence GFP fusion tagging.
低表达和分离过程中的不稳定性是阻碍充分进行膜蛋白（MPs）结构-功能研究的主要障碍。为了增加生成大量蛋白质的可能性，通常将 C 端融合的绿色荧光蛋白（GFP）用作监测表达和评估纯化的报告蛋白。由于 GFP 无法在大肠杆菌周质中发出荧光，该技术主要局限于具有细胞内 C 末端（C（in））的 MPs。借助于单个跨膜蛋白糖蛋白 A，我们开发了一种将具有细胞外 C 末端（C（out））的 MPs 转化为 C（in）的方法，为实施 GFP 报告提供了途径。我们在具有预测的 C（out）拓扑结构的 11 个 MPs 上测试了该方法，结果得到了高水平的表达。对于这 11 个 MPs 中的 9 个，使用扩展的荧光检测凝胶过滤色谱程序鉴定了稳定的、单分散的蛋白-去污剂复合物，该程序可随时间监测蛋白稳定性，这是影响结构-功能研究成功的关键参数。通过特异性蛋白酶切，成功地从 GFP 标签上切割了 5 个 MPs，并将其纯化至均一状态。为了解决蛋白水解效率低下的问题，我们在没有融合标签的情况下探索了表达和纯化条件。与之前的研究相反，融合标签建立的最佳表达条件不能直接转移到没有 GFP 标签的过表达中。这些研究建立了一种广泛适用于具有 C（out）拓扑结构的 MPs 的 GFP 筛选方法，产生了足够适合结构-功能研究的蛋白质，优于在没有 GFP 融合标签的情况下进行表达和纯化。
