Nedd4 is an E3 ubiquitin ligase that has received increased attention due to its role in the maintenance of proteostasis and in cellular stress responses. Investigation of Nedd4 enzymology has revealed a complex enzymatic mechanism that involves intermolecular interactions with upstream E2 conjugating enzymes and with substrates and intramolecular interactions that serve to regulate Nedd4 function. Thus, it is imperative that investigations of Nedd4 enzymology that employ recombinant enzyme be conducted with Nedd4 in its native, untagged form. We report herein an optimized, facile method for purification of recombinant human Nedd4 in its full-length form as a stable and active recombinant enzyme. Specifically, Nedd4 can be purified through a two-step purification which employs glutathione-S-transferase and hexahistidine sequences as orthogonal affinity tags. Proteolytic cleavage of Nedd4 was optimized to enable removal of the affinity tags with TEV protease, providing access to the untagged enzyme in yields of 2-3 mg/L. Additionally, investigation of Nedd4 storage conditions reveal that the enzyme is not stable through freeze-thaw cycles, and storage conditions should be carefully considered for preservation of enzyme stability. Finally, Nedd4 activity was validated through three activity assays which measure ubiquitin chain formation, Nedd4 autoubiquitination, and monoubiquitin consumption, respectively. Comparison of the method described herein with previously reported purification methods reveal that our optimized purification strategy enables access to Nedd4 in fewer chromatographic steps and eliminates reagents and materials that are potentially cost-prohibitive. This method, therefore, is more efficient and provides a more accessible route for purifying recombinant full-length Nedd4.

中文翻译：

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作为来自大肠杆菌的重组蛋白，对全长、未标记的人 Nedd4 进行稳健且简便的纯化。

Nedd4 是一种 E3 泛素连接酶，由于其在维持蛋白质稳态和细胞应激反应中的作用而受到越来越多的关注。对 Nedd4 酶学的研究揭示了一种复杂的酶促机制，该机制涉及与上游 E2 结合酶的分子间相互作用以及与用于调节 Nedd4 功能的底物和分子内相互作用。因此，必须对采用重组酶的 Nedd4 酶学进行研究，并以其天然的、未标记的形式对 Nedd4 进行研究。我们在此报告了一种优化的简便方法，用于纯化重组人 Nedd4 的全长形式，作为一种稳定且有活性的重组酶。具体来说，Nedd4 可以通过使用谷胱甘肽-S-转移酶和六组氨酸序列作为正交亲和标签的两步纯化来纯化。Nedd4 的蛋白水解裂解经过优化，可以去除带有 TEV 蛋白酶的亲和标签，从而以 2-3 mg/L 的产量提供未标记的酶。此外，对 Nedd4 储存条件的调查表明，该酶在冻融循环中不稳定，应仔细考虑储存条件以保持酶的稳定性。最后，通过分别测量泛素链形成、Nedd4 自泛素化和单泛素消耗的三种活性测定验证了 Nedd4 活性。将本文描述的方法与先前报道的纯化方法进行比较表明，我们优化的纯化策略能够以更少的色谱步骤获得 Nedd4，并消除了可能成本过高的试剂和材料。因此，该方法更有效，并为纯化重组全长 Nedd4 提供了更容易获得的途径。