The Strep-tag system for one-step purification and high-affinity detection or capturing of proteins
The Strep-tag system for one-step purification and high-affinity detection or capturing of proteins
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DOI：
10.1038/nprot.2007.209
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发表时间：
2007-01-01
期刊：
影响因子：
14.8
通讯作者：
Skerra, Arne
中科院分区：
文献类型：
--
作者：
Schmidt, Thomas G. M.;Skerra, Arne
Strep标签II是一个8个残基的最小肽序列（Trp-Ser-His-Pro-Gln-Phe-Glu-Lys），对链霉亲和素表现出固有的亲和力，可以以各种方式与重组蛋白融合。我们描述了一个协议，使快速和温和的纯化相应的Strep标签II融合蛋白-包括它们的复合物与相互作用的合作伙伴-无论是从细菌和真核细胞裂解物使用亲和层析的基质上进行工程链霉亲和素（Strep-Tactin），这可以在1小时内完成。高亲和力单克隆抗体（StrepMAB-Immo）允许Strep-标签II融合蛋白稳定固定到固体表面，例如用于表面等离子体共振分析。使用Strep-Tactin/酶偶联物或另一种单克隆抗体（StrepMAB-Classic）实现蛋白质印迹的选择性和灵敏度检测。因此，Strep-标签II，这是短的，生物惰性的，蛋白水解稳定，不干扰膜易位或蛋白质折叠，提供了一个通用的工具，用于快速分离的功能基因产物，并为它的检测或分子相互作用分析。
The Strep-tag II is an eight-residue minimal peptide sequence (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys) that exhibits intrinsic affinity toward streptavidin and can be fused to recombinant proteins in various fashions. We describe a protocol that enables quick and mild purification of corresponding Strep-tag II fusion proteins - including their complexes with interacting partners - both from bacterial and eukaryotic cell lysates using affinity chromatography on a matrix carrying an engineered streptavidin (Strep-Tactin), which can be accomplished within 1 h. A high- affinity monoclonal antibody (StrepMAB-Immo) permits stable immobilization of Strep-tag II fusion proteins to solid surfaces, for example, for surface plasmon resonance analysis. Selective and sensitive detection on western blots is achieved with Strep-Tactin/enzyme conjugates or another monoclonal antibody (StrepMAB-Classic). Thus, the Strep-tag II, which is short, biologically inert, proteolytically stable and does not interfere with membrane translocation or protein folding, offers a versatile tool both for the rapid isolation of a functional gene product and for its detection or molecular interaction analysis.