关键词： E1 enzyme E2 enzyme FAT10 Proteasome Protein conjugation Protein degradation Recombinant protein expression UBD Ubiquitin D Ubiquitin-like modifier

Mesh ： Blotting, Western Cell Line Gene Expression Humans Immunoprecipitation Proteasome Endopeptidase Complex / metabolism Proteolysis Recombinant Proteins / genetics isolation & purification metabolism Transfection / methods Ubiquitins / genetics isolation & purification metabolism Up-Regulation

来  源：   DOI：10.1016/bs.mie.2018.12.040   PDF(Sci-hub)

Abstract：
The ubiquitin-like modifier FAT10 (also called ubiquitin D (UBD)) interacts noncovalently with a substantial number of proteins and also gets covalently conjugated to many substrate proteins, leading to their degradation by the 26S proteasome. FAT10 comprises two loosely folded ubiquitin-like domains that are connected by a flexible linker, and this unusual structure makes it highly prone to aggregation. Here, we report methods to purify high amounts of soluble recombinant FAT10 for various uses, such as in vitro FAT10ylation assays. In addition, we describe how to generate and handle overexpressed as well as endogenous FAT10 in cellulo for use in immunoprecipitations, Western blot analyses, and FAT10 degradation studies.
摘要：
泛素样修饰剂FAT10（也称为泛素D（UBD））与大量蛋白质非共价相互作用，并与许多底物蛋白共价缀合，导致它们被26S蛋白酶体降解。FAT10包含两个通过柔性接头连接的松散折叠的泛素样结构域,这种不寻常的结构使它非常容易聚集。这里,我们报道了纯化大量可溶性重组FAT10用于各种用途的方法，如体外FAT10酰化测定。此外,我们描述了如何在细胞中产生和处理过表达的以及内源性FAT10，用于免疫沉淀，蛋白质印迹分析,和FAT10降解研究。
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