Intrinsically disordered proteins (IDPs) are subject to post-translational modifications. This allows the same polypeptide to be involved in different interaction networks with different consequences, ranging from regulatory signalling networks to the formation of membrane-less organelles. We report a robust method for co-expression of modification enzyme and SUMO-tagged IDPs with a subsequent purification procedure that allows for the production of modified IDP. The robustness of our protocol is demonstrated using a challenging system: RNA polymerase II C-terminal domain (CTD); that is, a low-complexity repetitive region with multiple phosphorylation sites. In vitro phosphorylation approaches fail to yield multiple-site phosphorylated CTD, whereas our in vivo protocol allows the rapid production of near homogeneous phosphorylated CTD at a low cost. These samples can be used in functional and structural studies.

中文翻译：

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高效，稳定地制备酪氨酸磷酸化的内在无序蛋白。

固有紊乱的蛋白质（IDP）受到翻译后修饰的影响。这允许同一多肽参与不同的相互作用网络，从而产生不同的后果，从调节信号网络到无膜细胞器的形成。我们报告了一种强大的方法，用于与修饰酶和SUMO标签的IDP共表达，并具有随后的纯化程序，可用于生产修饰的IDP。使用具有挑战性的系统可以证明我们协议的鲁棒性：RNA聚合酶II C末端域（CTD）；也就是说，具有多个磷酸化位点的低复杂度重复区域。体外磷酸化方法无法产生多位磷酸化CTD，而我们的体内协议允许以低成本快速生产接近均质的磷酸化CTD。这些样品可用于功能和结构研究。