Schmidt Thomas G M, Batz Lilia, Bonet Lidia, Carl Uwe, Holzapfel Gerd, Kiem Klaus, Matulewicz Kamila, Niermeier Dennis, Schuchardt Isabel, Stanar Kristian
IBA GmbH, Rudolf-Wissell-Str. 28, D-37079 Göttingen, Germany.
Protein Expr Purif. 2013 Nov;92(1):54-61. doi: 10.1016/j.pep.2013.08.021. Epub 2013 Sep 6.
Short peptide affinity tags have become indispensable in protein research. They cannot only be used for affinity purification but also downstream for detection and assay of an arbitrary fused recombinant protein without the need for any prior knowledge of its biochemical properties. Strep-tag®II is particularly popular for providing recombinant proteins at high purity and functionality by using physiological conditions within a rapid one-step protocol. The affinity receptor for Strep-tag®II is affinity engineered streptavidin, named Strep-Tactin®. Strep-tag®II binds to the biotin binding pocket enabling mild competitive elution with biotin derivatives, preferably desthiobiotin, for repeated use of the Strep-Tactin® affinity resins. Fast binding and dissociation kinetics allow comparatively high flow rates throughout column chromatography including elution. Fast dissociation kinetics may be, however, limiting for using Strep-tag®II for direct purification of target proteins from large volumes of diluted extracts like mammalian cell culture supernatants or in assay formats requiring extended washing like ELISA. For this reason, binding characteristics were improved by development of the Twin-Strep-tag® consisting of two Strep-tag®II moieties connected by a short linker. The resulting avidity effect, i.e., the combined synergistic binding of two Strep-tag®II moieties to tetrameric Strep-Tactin®, reduces the off-rate for more steady binding under non-competitive conditions. The addition of a competitor, however, reverses the synergistic avidity effect and, hence, efficient elution capability is preserved. In fact, the Twin-Strep-tag® features all beneficial properties of Strep-tag®II, including efficient elution under gentle competitive conditions, but, due to its higher affinity, additionally enables a more universal use in applications requiring stable binding.
短肽亲和标签在蛋白质研究中已变得不可或缺。它们不仅可用于亲和纯化，还可用于下游对任意融合重组蛋白的检测和分析，而无需事先了解其生化特性。链霉亲和素标签II（Strep-tag®II）通过在快速一步法方案中使用生理条件，以高纯度和功能性提供重组蛋白，因而特别受欢迎。链霉亲和素标签II的亲和受体是经过亲和工程改造的链霉抗生物素蛋白，称为链霉亲和素（Strep-Tactin®）。链霉亲和素标签II与生物素结合口袋结合，能够与生物素衍生物（最好是脱硫生物素）进行温和的竞争性洗脱，以便链霉亲和素亲和树脂能够重复使用。快速的结合和解离动力学使得在包括洗脱在内的整个柱色谱过程中能够采用相对较高的流速。然而，快速解离动力学可能会限制链霉亲和素标签II用于从大量稀释提取物（如哺乳动物细胞培养上清液）中直接纯化目标蛋白，或用于需要长时间洗涤的分析形式（如酶联免疫吸附测定）。因此，通过开发由两个通过短连接子连接的链霉亲和素标签II部分组成的双标签（Twin-Strep-tag®），改善了结合特性。由此产生的亲和力效应，即两个链霉亲和素标签II部分与四聚体链霉亲和素的协同结合，降低了解离速率，从而在非竞争性条件下实现更稳定的结合。然而，加入竞争者会逆转协同亲和力效应，因此保留了有效的洗脱能力。事实上，双标签具有链霉亲和素标签II所有的有益特性，包括在温和竞争条件下的有效洗脱，但由于其更高的亲和力，还能够在需要稳定结合的应用中更广泛地使用。
