Flies Andrew S, Darby Jocelyn M, Murphy Peter R, Pinfold Terry L, Patchett Amanda L, Lennard Patrick R
Menzies Institute for Medical Research, College of Health and Medicine, University of Tasmania, Hobart, TAS 7000, Australia.
University of Queensland Diamantina Institute, The University of Queensland, Translational Research Institute, Woolloongabba, Queensland, Australia.
Bio Protoc. 2020 Jul 5;10(13):e3696. doi: 10.21769/BioProtoc.3696.
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 parts: (A) characterization of the gene-of-interest (GOI) and protein-of-interest (POI); (B) design of the expression vector; (C) assembly of the expression vector; (D) transfection of a eukaryotic cell line with the expression vector; (E) testing of the recombinant protein. This extensive protocol can be completed with only polymerase chain reaction (PCR) and cell culture training. Additionally, each part of the protocol can be used independently.
本方案提供了一种逐步创建可从哺乳动物细胞系分泌的重组荧光融合蛋白的方法。这建立在许多其他重组蛋白和荧光蛋白技术的基础上，但却是最早利用直接分泌到细胞培养上清液中的荧光融合蛋白的方法之一。这开启了细菌或酵母中产生的蛋白质无法实现的新可能性，例如在活细胞共培养试验中直接使用分泌荧光蛋白的细胞。荧光适应性简易治疗诊断（FAST）蛋白系统包括一个组氨酸纯化标签和一个烟草蚀纹病毒（TEV）切割位点，可去除纯化标签和荧光蛋白以用于治疗。本方案分为五个部分：（A）目的基因（GOI）和目的蛋白（POI）的表征；（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）重组蛋白的检测。仅通过聚合酶链反应（PCR）和细胞培养训练就能完成这个详尽的方案。此外，该方案的每个部分都可独立使用。
