VIB-VUB Center for Structural Biology, Vlaams Instituut voor Biotechnologie (VIB), 1050 Brussels, Belgium.
Structural Biology Brussels (SBB), Vrije Universiteit Brussel (VUB), 1050 Brussels, Belgium.
Biomolecules. 2022 Oct 26;12(11):1566. doi: 10.3390/biom12111566.
Intrinsically disordered proteins (IDPs) lack well-defined 3D structures and can only be described as ensembles of different conformations. This high degree of flexibility allows them to interact promiscuously and makes them capable of fulfilling unique and versatile regulatory roles in cellular processes. These functional benefits make IDPs widespread in nature, existing in every living organism from bacteria and fungi to plants and animals. Due to their open and exposed structural state, IDPs are much more prone to proteolytic degradation than their globular counterparts. Therefore, the purification of recombinant IDPs requires extra care and caution, such as maintaining low temperature throughout the purification, the use of protease inhibitor cocktails and fast workflow. Even so, in the case of long IDP targets, the appearance of truncated by-products often seems unavoidable. The separation of these unwanted proteins can be very challenging due to their similarity to the parent target protein. Here, we describe a tandem-tag purification method that offers a remedy to this problem. It contains only common affinity-chromatography steps (HisTrap and Heparin) to ensure low cost, easy access and scaling-up for possible industrial use. The effectiveness of the method is demonstrated with four examples, Tau-441 and two of its fragments and the transactivation domain (AF1) of androgen receptor.
无规卷曲蛋白质（IDPs）缺乏明确的 3D 结构，只能被描述为不同构象的集合。这种高度的灵活性使它们能够随意相互作用，使它们能够在细胞过程中发挥独特而多样的调节作用。这些功能上的好处使得 IDPs 在自然界中广泛存在，从细菌和真菌到植物和动物，每种生物中都存在 IDPs。由于其开放和暴露的结构状态，IDPs 比其球状对应物更容易受到蛋白水解降解。因此，重组 IDPs 的纯化需要格外小心和谨慎，例如在整个纯化过程中保持低温、使用蛋白酶抑制剂混合物和快速工作流程。即便如此，对于长的 IDP 靶标，截断副产物的出现似乎仍然不可避免。由于这些不需要的蛋白质与母体靶蛋白相似，因此分离它们非常具有挑战性。在这里，我们描述了一种串联标签纯化方法，为解决这个问题提供了一种补救措施。它仅包含常见的亲和层析步骤（HisTrap 和肝素），以确保低成本、易于获得和可能用于工业用途的规模化。该方法的有效性通过四个例子得到了证明，即 Tau-441 和它的两个片段以及雄激素受体的转录激活结构域（AF1）。
