Engineering and bioconjugation of proteins is a critically valuable tool that can facilitate a wide range of biophysical and structural studies. The ability to orthogonally tag or label a domain within a multidomain protein may be complicated by undesirable side reactions to noninvolved domains. Furthermore, the advantages of segmental (or domain-specific) isotopic labeling for NMR, or deuteration for neutron scattering or diffraction, can be realized by an efficient ligation procedure. Common methods-expressed protein ligation, protein trans-splicing, and native chemical ligation-each have specific limitations. Here, we evaluated the use of different variants of Staphylococcus aureus sortase A for a range of ligation reactions and demonstrate that conditions can readily be optimized to yield high efficiency (i.e. completeness of ligation), ease of purification, and functionality in detergents. These properties may enable joining of single domains into multidomain proteins, lipidation to mimic posttranslational modifications, and formation of cyclic proteins to aid in the development of nanodisc membrane mimetics. We anticipate that the method for ligating separate domains into a single functional multidomain protein reported here may enable many applications in structural biology.

中文翻译：

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分拣酶A连接的优化，可灵活构建复杂的蛋白质系统。

蛋白质的工程和生物缀合是至关重要的工具，可以促进广泛的生物物理和结构研究。正交标记或标记多结构域蛋白质中的结构域的能力可能会由于与非参与结构域发生的不良副反应而变得复杂。此外，可以通过有效的连接程序来实现针对NMR进行分段（或域特定）同位素标记或对中子散射或衍射进行氘化的优势。表达蛋白质的连接，蛋白质反式剪接和天然化学连接的常用方法都有特定的局限性。在这里，我们评估了金黄色葡萄球菌分选酶A的不同变体在一系列连接反应中的使用，并证明可以轻松优化条件以产生高效率（即连接的完整性），易于纯化，并具有清洁剂的功能。这些性质可以使单结构域结合到多结构域蛋白中，脂化以模仿翻译后修饰，以及形成环状蛋白以帮助开发纳米盘膜模拟物。我们预计，将单独的结构域连接到此处报道的单个功能性多结构域蛋白的方法可能会在结构生物学中实现许多应用。