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Author:
Date:
2020-07-05
[Abstract] This protocol provides a step-by-step method to create recombinant fluorescent fusion proteins that can be secreted from mammalian cell lines. This builds on many other recombinant protein and fluorescent protein techniques, but is among the first to harness fluorescent fusion proteins secreted directly into cell culture supernatant. This opens new possibilities that are not achievable with proteins produced in bacteria or yeast, such as direct use of the fluorescent protein-secreting cells in live co-culture assays. The Fluorescent Adaptable Simple Theranostic (FAST) protein system includes a histidine purification tag and a tobacco etch virus (TEV) cleavage site, allowing the purification tag and fluorescent protein to be removed for therapeutic use. This protocol is split into five ...
[摘要] [摘要] 该方案提供了一种逐步建立重组荧光融合蛋白的方法，可从哺乳动物细胞系分泌。这项技术建立在许多其他重组蛋白和荧光蛋白技术的基础上，但它是第一个利用荧光融合蛋白直接分泌到细胞培养上清液中的技术之一。这为细菌或酵母产生的蛋白质开辟了新的可能性，例如直接使用荧光蛋白分泌细胞进行活体共培养实验。该荧光适应性简单热（FAST）蛋白系统包括组氨酸纯化标签和烟草蚀刻病毒（TEV）裂解位点，允许去除纯化标签和荧光蛋白用于治疗用途。该方案分为五个部分：（A）感兴趣基因（GOI）和感兴趣蛋白（POI）的电子表征；（B）表达载体的设计；（C）表达载体的组装；（D）用表达载体转染真核细胞系；（E）检测重组蛋白。这种广泛的方案只需聚合酶链反应（PCR）和细胞培养训练即可完成。另外，协议的每个部分都可以独立使用。
[背景] ...
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