Purification of protein complexes of defined subunit stoichiometry using a set of orthogonal, tag-cleaving proteases
Purification of protein complexes of defined subunit stoichiometry using a set of orthogonal, tag-cleaving proteases
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DOI：
10.1016/j.chroma.2014.02.030
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发表时间：
2014-04-11
影响因子：
4.1
通讯作者：
Goerlich, Dirk
中科院分区：
文献类型：
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作者：
Frey, Steffen;Goerlich, Dirk
无标签蛋白质或蛋白质复合物无疑代表了功能或结构研究最真实的起点。它们可以通过传统的多步色谱法从天然或重组的无标签来源获得。或者，它们可以使用可切割的 N 端亲和标签进行表达和纯化，随后通过位点特异性蛋白酶将其去除。蛋白水解标签去除也可以在“柱上”进行。我们在此表明，这不仅代表了非常有效的工作流程，而且还大大提高了所得蛋白质制剂的纯度。然而，有效柱上切割的先决条件是标签切割蛋白酶不结合固定相。我们介绍 scAtg4 和 xlUsp2 非常好，bdSENP1、bdNEDP1 以及 ssNEDP1 作为 4 摄氏度柱上裂解的理想蛋白酶。其中四种蛋白酶（bdSENP1、bdNEDP1、scAtg4、xlUsp2）以及 TEV 蛋白酶显示正交，即互斥的裂解特异性。我们将这些特征组合成一种生产高纯度蛋白质复合物的简化方法：正交亲和标签和蛋白酶识别模块融合到各个亚基上。在共表达或体外复合物组装之后，亲和捕获和蛋白水解释放的连续循环然后依次选择每个正交标记的亚基的存在，产生明确的亚基化学计量的蛋白质复合物。 (C) 2014 年作者。由 Elsevier B.V. 出版
Tag-free proteins or protein complexes represent certainly the most authentic starting points for functional or structural studies. They can be obtained by conventional multi-step chromatography from native or recombinant tag-free sources. Alternatively, they can be expressed and purified using a cleavable N-terminal affinity tag that is subsequently removed by a site-specific protease. Proteolytic tag-removal can also be performed "on-column". We show here that this not only represents a very efficient workflow, but also drastically improves the purity of the resulting protein preparations. Precondition for effective on-column-cleavage is, however, that the tag-cleaving protease does not bind the stationary phase. We introduce scAtg4 and xlUsp2 as very good and bdSENP1, bdNEDP1 as well as ssNEDP1 as ideal proteases for on-column cleavage at 4 degrees C. Four of these proteases (bdSENP1, bdNEDP1, scAtg4, xlUsp2) as well as TEV protease display orthogonal, i.e. mutually exclusive cleavage specificities. We combined these features into a streamlined method for the production of highly pure protein complexes: Orthogonal affinity tags and protease recognitions modules are fused to individual subunits. Following co-expression or in-vitro complex assembly, consecutive cycles of affinity capture and proteolytic release then select sequentially for the presence of each orthogonally tagged subunit, yielding protein complexes of well-defined subunit stoichiometry. (C) 2014 The Authors. Published by Elsevier B.V.