Next generation calmodulin affinity purification: Clickable calmodulin facilitates improved protein purification.
Next generation calmodulin affinity purification: Clickable calmodulin facilitates improved protein purification.
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DOI：
10.1371/journal.pone.0197120
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发表时间：
2018
期刊：
影响因子：
3.7
通讯作者：
Kinzer-Ursem TL
中科院分区：
文献类型：
--
作者：
Fraseur JG;Kinzer-Ursem TL
随着蛋白质组学领域的不断扩大，科学家们正在寻求整合跨学科的工具来研究蛋白质的结构，功能和相互作用。蛋白质纯化仍然是许多表征研究的关键工具。钙调素（CaM）是一种钙结合信使蛋白，具有超过100个下游结合伴侣，并且参与许多生理过程，从学习和记忆到免疫和心脏功能。为了促进钙调素的生物物理研究，研究人员设计了一种用于生物缀合应用的位点特异性标记方法，同时保持高水平的蛋白质活性。在这里，我们提出了一个平台，直接从澄清的细胞裂解液在生物正交反应条件下的钙调蛋白的选择性共轭。使用化学酶修饰的钙调蛋白，我们采用流行的点击化学反应的钙调蛋白的琼脂糖凝胶树脂的共轭，从而简化了以前的多步纯化和共轭过程。我们表明，这种“下一代”钙调素-琼脂糖凝胶树脂不仅易于生产，而且还能够纯化比传统钙调素-琼脂糖凝胶树脂更多的钙调素结合蛋白。我们希望这些方法可以转化为其他感兴趣的蛋白质和其他缀合应用，如基于表面的蛋白质-蛋白质相互作用动力学表征的测定。
As the proteomics field continues to expand, scientists are looking to integrate cross-disciplinary tools for studying protein structure, function, and interactions. Protein purification remains a key tool for many characterization studies. Calmodulin (CaM) is a calcium-binding messenger protein with over a hundred downstream binding partners, and is involved in a host of physiological processes, from learning and memory to immune and cardiac function. To facilitate biophysical studies of calmodulin, researchers have designed a site-specific labeling process for use in bioconjugation applications while maintaining high levels of protein activity. Here, we present a platform for selective conjugation of calmodulin directly from clarified cell lysates under bioorthogonal reaction conditions. Using a chemoenzymatically modified calmodulin, we employ popular click chemistry reactions for the conjugation of calmodulin to Sepharose resin, thereby streamlining a previously multi-step purification and conjugation process. We show that this “next-generation” calmodulin-Sepharose resin is not only easy to produce, but is also able to purify more calmodulin-binding proteins per volume of resin than traditional calmodulin-Sepharose resins. We expect these methods to be translatable to other proteins of interest and to other conjugation applications such as surface-based assays for the characterization of protein-protein interaction dynamics.
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