Production of Eukaryotic Glycoproteins for Structural and Functional Studies Using Expi293F Cells.
Production of Eukaryotic Glycoproteins for Structural and Functional Studies Using Expi293F Cells.
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DOI：
10.1002/cpz1.512
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发表时间：
2022-08
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需要毫克量的纯蛋白质进行结构、功能和药物筛选研究。这些要求对于大多数重要的治疗靶点是具有挑战性的，所述治疗靶点是分泌的糖蛋白、受体、膜蛋白或大的胞质复合物。在这里，我们提出了一个协议，用于生产和纯化大量的分泌糖蛋白使用哺乳动物细胞为基础的Expi293F系统通过大规模的瞬时转染。该系统可以很容易地适用于膜蛋白和大的胞质复合物的生产。该方法可用于快速评估许多表达构建体以鉴定最佳表达体。使用哺乳动物细胞确保了正确的翻译后修饰，包括二硫键和糖基化，这对于准确的功能研究非常重要。此外，可以引入微小的修饰以产生标记的或去糖基化的蛋白质，用于通过X射线晶体学、NMR或冷冻电子显微镜进行结构研究。基本方案1：基本方案2：Expi293F细胞的大规模培养和瞬时转染基本方案3：从培养基中纯化六组氨酸标记的蛋白质
Milligram quantities of pure proteins are required for structural, functional, and pharmaceutical screening studies. These requirements can be challenging for a majority of important therapeutic targets that are secreted glycoproteins, receptors, membrane proteins, or large cytosolic complexes. Here we present a protocol for producing and purifying large amounts of secreted glycoproteins using the mammalian cell-based Expi293F system via large-scale transient transfection. This system can be easily adapted for the production of membrane proteins and large cytosolic complexes. The method can be utilized to quickly evaluate numerous expression constructs to identify optimal expressers. The use of mammalian cells ensures proper postranslational modifications, including disulfide bonds and glycosylation, that can be important for accurate functional studies. In addition, minor modifications can be introduced to produce labeled or deglycosylated proteins for structural studies by X-ray crystallography, NMR, or cryo-electron microscopy. Basic Protocol 1: Production of milligram quantities of plasmid DNA for large-scale transient transfection Basic Protocol 2: Large-scale culture and transient transfection of Expi293F cells Basic Protocol 3: Purification of hexahistidine-tagged proteins from media