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细说小分子短肽标签（三）之Strep-Tag II

发布: 2016-02-03 1,567阅读 162评论

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。

第三篇文章要介绍的是同样使用很普遍的短肽亲和标签-Strep-Tag II。跟His-Tag和FLAG-Tag相似，它已经被广泛应用于大肠杆菌，酵母和哺乳动物细胞等表达系统中。Strep-tag II由8个氨基酸残基组成，序列是Trp-Ser-His-Pro-Gln-Phe-Glu-Lys（W-S-H-P-Q-F-E-K）。因为它的分子量很小，只有1.06kDa，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。

b

在这个亲和标签的发现和设计上，科学家还走过一些“弯路”。最初，他们发现了自然界中链霉亲和素 （streptavidin）和生物素（biotin）具有极强烈的非共价相互作用（non-covalent interactions），而且生物学上的解离常数（dissociation constant）非常低，数量级在10−14 mol/L左右；即使生物素和其他蛋白质共价结合之后，也不影响它和链霉亲和素的相互作用。基于这两者之间强烈的相互作用，科学家运用一系列蛋白质工程手段，通过对短肽文库的筛选，设计了第一个链霉亲和素结合肽标签-Strep-tag。但是Strep-Tag和链霉亲和素结合时需要一个自由的羧基末端，所以这个标签只能位于目标蛋白的Ｃ端，很大的限制了它的应用范围。在第一代标签的基础上，通过大量合成短肽的筛选，终于获得了一个和一代相似、也是由８个氨基酸（W-S-H-P-Q-F-E-K）组成的链霉亲和素结合肽，就是Strep-Tag II，它可以位于融合蛋白的任意一端，弥补了最初的不足 (Terpe, 2003)。

不但如此，科学家们还通过蛋白质工程工程手段改进了链霉亲和素的蛋白序列，取名为Strep-Tactin。如此一来，Strep-tag与Strep-Tactin的结合力比天然的链霉亲和素要高至少100倍。这个亲和标签系统的纯化条件比较宽泛，不但在普通缓冲液（例如磷酸盐缓冲溶液， PBS）下可以和Strep-Tactin层析介质（resin）结合，而且使用2.5mM的脱硫生物素 （desthiobiotin）就可快速的将融合蛋白洗脱下来。溶液中如果带有螯合剂、去污剂、还原剂及高达1M的盐等等，也不会影响Strep-Tag II系统的效果。图一清晰明了的展示了使用Strep-Tag II 和Strep-Tactin提纯蛋白的过程。

c

图1：使用Strep-Tag II 系统蛋白提纯示意图

从左到右：

1. 新的或是重新利用的Strep-Tactin 层析介质；

2. 带有Strep-Tag II 的绿色荧光蛋白（GFP）和层析介质的结合，其他的蛋白杂质将会被缓冲液冲洗掉；

3. 加入脱硫生物素后，将目标蛋白洗脱；

4-6. 层析介质柱的再利用的过程: 脱硫生物素会被黄色溶液HABA移除；一旦柱子变红，证明HABA与Strep-Tactin结合。用缓冲液再将HABA溶液去除，层析介质柱就可以重复使用。

蛋白提纯的整个过程简单又方便，而且检测的方法也多种多样。除了有针对Strep-Tag II的特异性抗体外，还有直接与HRP或是AP连结的Strep-Tactin。例如，在Western Blot中，通过直接法（direct method），就可以检测到目标蛋白的表达。图二就是这个实验过程的简易图，便于大家理解。不但如此，科学家还设计了Twin-Strep-Tag，进一步加强对目标蛋白检测的敏感度和定量的准确性。这个标签由30个氨基酸残基组成，序列是S-A-W-S-H-P-Q-F-E-K-G-G-G-S)2-G-G-S-A-W-S-H-P-Q-F-E-K。根据实验表明，仅一步提纯后的蛋白纯度就能达到99%。标签虽好，但毕竟这是一个分子量较大的蛋白残基，有可能会影响目标蛋白的溶解度或是特性，不一定适用所有表达系统或是目标蛋白。所以大家无论设计什么标签，都要根据自己的目标蛋白和实验目的出发。

d

图2：用Strep-Tactin检测目标蛋白示意图

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】http://www.iba-lifesciences.com/strep-tag.html

【3】http://www.iba-lifesciences.com/twin-strep-tag.html

【4】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

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  161. 先赞一个 通篇看完一字不漏 基本了解了亲和标签蛋白纯化

    请问一下
    如果想详细了解标签亲和纯化可以看哪些教材或者参考书？谢谢。

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    1. 可以看看这本教材，里面有所介绍。我当时读书时的教材。生物分离工程(孙彦)。

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