Hill Spencer, Hill Connor, Kleiger Gary
Department of Chemistry and Biochemistry, University of Nevada, Las Vegas, NV, USA.
Methods Mol Biol. 2018;1844:39-58. doi: 10.1007/978-1-4939-8706-1_4.
Ubiquitin ligases (E3s) function by binding to both a protein substrate and to ubiquitin-conjugating enzymes (E2s) bound to ubiquitin. E3s facilitate the transfer of ubiquitin from the E2 active site to an E3-bound substrate. Thus, the affinity of the interaction of an E2 with its E3 partner is of considerable interest. The purpose of this work is to (1) provide protocols for the purification of the human E2 Cdc34, as well as for some additional protein components needed for the assays described here whose purification protocols haven't been described elsewhere in detail; (2) provide the researcher with critical information regarding the proper long-term storage of these enzymes to retain maximal activity; (3) provide a protocol to benchmark Cdc34 activity with previously described activity levels in the literature; and (4) provide a simple and rapid means of measuring E2 affinity for an E3.
泛素连接酶（E3s）通过与蛋白质底物以及与结合了泛素的泛素结合酶（E2s）结合来发挥作用。E3s促进泛素从E2活性位点转移至与E3结合的底物上。因此，E2与其E3伴侣之间相互作用的亲和力备受关注。本研究的目的是：（1）提供纯化人E2 Cdc34的方案，以及提供此处所述实验所需的一些其他蛋白质成分的纯化方案，这些成分的纯化方案在其他地方尚未详细描述；（2）为研究人员提供有关这些酶适当长期储存以保持最大活性的关键信息；（3）提供一个方案，以便根据文献中先前描述的活性水平来衡量Cdc34的活性；（4）提供一种简单快速的方法来测量E2对E3的亲和力。
