Ohana Rachel Friedman, Hurst Robin, Vidugiriene Jolanta, Slater Michael R, Wood Keith V, Urh Marjeta
Promega Corporation, Madison, WI 53711, USA.
Protein Expr Purif. 2011 Apr;76(2):154-64. doi: 10.1016/j.pep.2010.11.014. Epub 2010 Dec 1.
Although cultured mammalian cells are preferred for producing functional mammalian proteins with appropriate post-translational modifications, purification of recombinant proteins is frequently hampered by low expression. We have addressed this by creating a new method configured specifically for mammalian cell culture that provides rapid detection and efficient purification. This approach is based on HaloTag, a protein fusion tag designed to bind rapidly, selectively and covalently to a series of synthetic ligands that can carry a variety of functional groups, including fluorescent dyes for detection or solid supports for purification. Since the binding of HaloTag to the HaloLink resin is essentially irreversible, it overcomes the equilibrium-based binding limitations associated with affinity tags and enables efficient capture and purification of target protein, even at low expression levels. The target protein is released from the HaloLink resin by specific cleavage using a TEV protease fused to HaloTag (HaloTEV), leaving both HaloTag and HaloTEV permanently attached to the resin and highly pure, tag-free protein in solution. HaloTag fluorescent ligands enable fluorescent labeling of HaloTag fusion proteins, providing a convenient way to monitor expression, and thus facilitate the identification of optimal transient transfection conditions as well as the selection of high expression stable cell lines. The capabilities of this method have been demonstrated by the efficient purification of five functional human kinases from HEK293T cells. In addition, when purifications using FLAG, 3xFLAG, His(6)Tag and HaloTag were performed in parallel, HaloTag was shown to provide significantly higher yields, purity and overall recovery of the expressed proteins.
尽管培养的哺乳动物细胞因能产生具有适当翻译后修饰的功能性哺乳动物蛋白而备受青睐，但重组蛋白的纯化常常因低表达而受阻。我们通过创建一种专门为哺乳动物细胞培养配置的新方法来解决这一问题，该方法可实现快速检测和高效纯化。这种方法基于HaloTag，一种蛋白质融合标签，它能快速、选择性且共价地结合一系列可携带多种功能基团的合成配体，这些功能基团包括用于检测的荧光染料或用于纯化的固体支持物。由于HaloTag与HaloLink树脂的结合基本上是不可逆的，它克服了与亲和标签相关的基于平衡的结合限制，即使在低表达水平下也能有效捕获和纯化目标蛋白。通过使用与HaloTag融合的TEV蛋白酶（HaloTEV）进行特异性切割，可将目标蛋白从HaloLink树脂上释放出来，使HaloTag和HaloTEV都永久附着在树脂上，而溶液中则是高度纯净的、无标签的蛋白。HaloTag荧光配体能够对HaloTag融合蛋白进行荧光标记，为监测表达提供了一种便捷的方式，从而有助于确定最佳的瞬时转染条件以及筛选高表达稳定细胞系。从HEK293T细胞中高效纯化五种功能性人类激酶，证明了该方法的能力。此外，当同时使用FLAG、3xFLAG、His(6)Tag和HaloTag进行纯化时，结果表明HaloTag能显著提高表达蛋白的产量、纯度和总体回收率。
