在分析蛋白质的结构和功能时，最重要的是如何得到高纯度的蛋白。现今最常用的方法就是亲和标签系统，已经成为重组蛋白检测和纯化中一个不可或缺的工具。不但如此，它还可能对目标蛋白的理化性质产生有利的影响，甚至可以提高重组蛋白的产量。亲和标签有多种不同的分类方法。根据分子量的大小，可分为“小分子短肽标签”和“蛋白质标签”两大类。小编特地为大家撰写了“细说小分子短肽标签”系列文章，希望能够帮助大家从众多蛋白标签中选择适合自己研究课题的。
第二篇文章要介绍的就是目前除了His-tag之外另一个使用很普遍的亲和标签-FLAG-tag。FLAG-tag被广泛应用于各种表达系统中, 例如大肠杆菌，酵母和哺乳动物细胞。FLAG-tag由8个氨基酸残基组成，序列是Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys （D-Y-K-D-D-D-D-K）。因为它的分子量很小，只有1.01kDa，所以不会遮盖融合蛋白中其他的蛋白表位和结构域，更不容易改变融合蛋白的功能、分泌或是运输。
标签序列中四个连着的天冬氨酸让整个标签具有天然的亲水特性（hydrophilicity）。这样一来，它就很容易定位在融合蛋白的表面，便于利用抗体来检测。值得注意的是，芳香环在抗原-抗体结合中具有很大的作用，所以这个标签的第二位残基特意设计的是酪氨酸（tyrosine）。因为FLAG-tag本身就具有免疫原型，所以它有３个特异性的单克隆抗体，分别是M1单抗，M2单抗，M5单抗。这就使得融合蛋白检测和提纯都变得很便利。但是实验表明FLAG-tag和特异性的抗体结合力没有His-tag和Ni2+-NTA或是其他一些亲和标签和检测系统的结合力那么牢固，融合蛋白的纯度只能达到90%左右 (Terpe,2003)。不过不要紧，大家还可以把三个FLAG-tag设计在一起（DYKDHDG-DYKDHDI-DYKDDDDK），增加和抗体的结合以及检测的敏感度。实验证明，3FLAG甚至能检测到飞量级的融合蛋白，比其他任何系统约高出20到200倍，特别适合于哺乳动物细胞表达系统中低表达水平蛋白质的检测 。
与此同时，FLAG-tag含有一个肠激酶（enterokinase）的切割位点（D-D-D-D-K-X1）。实验证明X1这个氨基酸残基是影响肠激酶的切割效率的。恰巧赖氨酸（Lysine）在X1的位置时，切割效率达到了85%，仅次于丙氨酸 （alanine）和甲硫氨酸 （methionine）(Einhauer & Jungbauer, 2001)。通常情况下，FLAG-tag被放置在N端，通过肠激酶切掉后，就会还原融合蛋白的原始序列和结构。
Reference:
【1】陈爱春, 彭伟, 汪生鹏 “亲和标签在重组蛋白表达与纯化中的应用” 中国生物工程杂志 China Biotechnology, 2012, 32 (12): 93-103
【2】Einhauer, A., & Jungbauer, A. (2001). The FLAG e peptide , a versatile fusion tag for the purification of recombinant proteins, 455–465
【3】Terpe, K. (2003). Overview of tag protein fusions : from molecular and biochemical fundamentals to commercial systems, 523–533. http://doi.org/10.1007/s00253-002-1158-6
【4】http://www.sigmaaldrich.com/life-science/proteomics/recombinant-protein-expression/purification-detection/flag-system.html
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