Department of Chemistry and Biochemistry, University of Maryland Baltimore County, Baltimore, MD, United States.
Department of Chemistry and Biochemistry, University of Maryland Baltimore County, Baltimore, MD, United States.
Methods Enzymol. 2023;679:235-254. doi: 10.1016/bs.mie.2022.07.036. Epub 2022 Aug 31.
Arginyltransferases (ATE1s) are eukaryotic enzymes that catalyze the non-ribosomal, post-translational addition of the amino acid arginine to an acceptor protein. While understudied, post-translation arginylation and ATE1 have major impacts on eukaryotic cellular homeostasis through both degradative and non-degradative effects on the intracellular proteome. Consequently, ATE1-catalyzed arginylation impacts major eukaryotic biological processes including the stress response, cellular motility, cardiovascular maturation, and even neurological function. Despite this importance, there is a lack of information on the structural and biophysical characteristics of ATE1, prohibiting a comprehensive understanding of the mechanism of this post-translational modification, and hampering efforts to design ATE1-specific therapeutics. To that end, this chapter details a protocol designed for the expression and the purification of ATE1 from Saccharomyces cerevisiae, although the approaches described herein should be generally applicable to other eukaryotic ATE1s. The detailed procedures afford high amounts of pure, homogeneous, monodisperse ATE1 suitable for downstream biophysical analyses such as X-ray crystallography, small angle X-ray scattering (SAXS), and cryo-EM techniques.
精氨酰基转移酶（ATE1s）是一类真核酶，能够在蛋白质翻译后将精氨酸非核糖体地添加到靶蛋白上。尽管研究较少，但通过对细胞内蛋白质组的降解和非降解作用，翻译后精氨酰化和 ATE1 对真核细胞的内稳态具有重大影响。因此，ATE1 催化的精氨酰化影响包括应激反应、细胞运动、心血管成熟甚至神经功能在内的主要真核生物过程。尽管具有重要意义，但对于 ATE1 的结构和生物物理特性的信息仍然缺乏，这阻碍了对这种翻译后修饰机制的全面理解，并妨碍了设计 ATE1 特异性治疗方法的努力。为此，本章详细介绍了从酿酒酵母中表达和纯化 ATE1 的方案，尽管本文所述的方法应普遍适用于其他真核 ATE1。详细的程序可提供大量高纯度、均一、单分散的 ATE1，适用于 X 射线晶体学、小角 X 射线散射（SAXS）和冷冻电镜技术等下游生物物理分析。
