Ivanov Konstantin I, Bašić Marta, Varjosalo Markku, Mäkinen Kristiina
Department of Food and Environmental Sciences, University of Helsinki.
Institute of Biotechnology, University of Helsinki.
J Vis Exp. 2014 Apr 20(86):51536. doi: 10.3791/51536.
Affinity purification of Strep-tagged fusion proteins on resins carrying an engineered streptavidin (Strep-Tactin) has become a widely used method for isolation of protein complexes under physiological conditions. Fusion proteins containing two copies of Strep-tag II, designated twin-Strep-tag or SIII-tag, have the advantage of higher affinity for Strep-Tactin compared to those containing only a single Strep-tag, thus allowing more efficient protein purification. However, this advantage is offset by the fact that elution of twin-Strep-tagged proteins with biotin may be incomplete, leading to low protein recovery. The recovery can be dramatically improved by using denaturing elution with sodium dodecyl sulfate (SDS), but this leads to sample contamination with Strep-Tactin released from the resin, making the assay incompatible with downstream proteomic analysis. To overcome this limitation, we have developed a method whereby resin-coupled tetramer of Strep-Tactin is first stabilized by covalent cross-linking with Bis(sulfosuccinimidyl) suberate (BS3) and the resulting cross-linked resin is then used to purify target protein complexes in a single batch purification step. Efficient elution with SDS ensures good protein recovery, while the absence of contaminating Strep-Tactin allows downstream protein analysis by mass spectrometry. As a proof of concept, we describe here a protocol for purification of SIII-tagged viral protein VPg-Pro from nuclei of virus-infected N. benthamiana plants using the Strep-Tactin polymethacrylate resin cross-linked with BS3. The same protocol can be used to purify any twin-Strep-tagged protein of interest and characterize its physiological binding partners.
利用携带工程化链霉抗生物素蛋白（链霉亲和素）的树脂对带有链霉标签的融合蛋白进行亲和纯化，已成为在生理条件下分离蛋白质复合物的一种广泛应用的方法。含有两个链霉标签II拷贝的融合蛋白，称为双连链霉标签或SIII标签，与仅含有单个链霉标签的融合蛋白相比，对链霉亲和素有更高的亲和力，因此能实现更高效的蛋白质纯化。然而，用生物素洗脱双连链霉标签蛋白可能不完全，导致蛋白质回收率低，这一缺点抵消了上述优势。使用十二烷基硫酸钠（SDS）进行变性洗脱可显著提高回收率，但这会导致样品被从树脂上释放的链霉亲和素污染，使得该检测方法与下游蛋白质组学分析不兼容。为克服这一局限性，我们开发了一种方法，即首先通过与双（磺基琥珀酰亚胺）辛二酸酯（BS3）共价交联来稳定树脂偶联的链霉亲和素四聚体，然后将所得的交联树脂用于在单一批次纯化步骤中纯化目标蛋白质复合物。用SDS进行高效洗脱可确保良好的蛋白质回收率，同时不存在污染性的链霉亲和素，从而可进行下游的质谱蛋白质分析。作为概念验证，我们在此描述了一种使用与BS3交联的链霉亲和素聚甲基丙烯酸酯树脂从病毒感染的本氏烟草植物细胞核中纯化SIII标签病毒蛋白VPg-Pro的方案。相同的方案可用于纯化任何感兴趣的双连链霉标签蛋白，并鉴定其生理结合伴侣。
