Lee Christine C, Li Bing, Yu Hongtao, Matunis Michael J
Department of Biochemistry & Molecular Biology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA.
Department of Pharmacology, Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX, USA.
Bio Protoc. 2018 Oct 5;8(19). doi: 10.21769/BioProtoc.3033.
The Small Ubiquitin-related Modifier (SUMO) is a protein that is post-translationally added to and reversibly removed from other proteins in eukaryotic cells. SUMO and enzymes of the SUMO pathway are well conserved from yeast to humans and SUMO modification regulates a variety of essential cellular processes including transcription, chromatin remodeling, DNA damage repair, and cell cycle progression. One of the challenges in studying SUMO modification is the relatively low steady-state level of a SUMO-modified protein due in part to the activity of SUMO deconjugating enzymes known as SUMO Isopeptidases or SENPs. Fortunately, the use of recombinant SUMO enzymes makes it possible to study SUMO modification . Here, we describe a sensitive method for detecting SUMO modification of target human proteins using an transcription and translation system derived from rabbit reticulocyte and radiolabeled amino acids.
小泛素相关修饰物（SUMO）是一种在真核细胞中通过翻译后修饰添加到其他蛋白质上并可从这些蛋白质上可逆去除的蛋白质。SUMO及SUMO途径中的酶从酵母到人类都高度保守，SUMO修饰调节多种重要的细胞过程，包括转录、染色质重塑、DNA损伤修复和细胞周期进程。研究SUMO修饰面临的挑战之一是SUMO修饰蛋白的稳态水平相对较低，部分原因是SUMO去共轭酶（称为SUMO异肽酶或SENP）的活性。幸运的是，重组SUMO酶的使用使得研究SUMO修饰成为可能。在此，我们描述了一种使用源自兔网织红细胞的转录和翻译系统及放射性标记氨基酸来检测目标人类蛋白质SUMO修饰的灵敏方法。
