TET 蛋白的纯化。

Purification of TET Proteins.

机构信息

Center for Epigenetics, Van Andel Institute, Grand Rapids, MI, USA.

Asan Medical Center, University of Ulsan, College of Medicine, Seoul, South Korea.

出版信息

Methods Mol Biol. 2021;2272:225-237. doi: 10.1007/978-1-0716-1294-1_13.

DOI:10.1007/978-1-0716-1294-1_13

PMID:34009617

Abstract

The 5-methylcytosine (5mC) oxidation pathway mediated by TET proteins involves step-wise oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). 5fC and 5caC can be removed from DNA by base excision repair and the completion of this pathway results in "demethylation" of 5mC by converting the modified base back into cytosine. In vitro studies with TET proteins aimed at analyzing their DNA substrate specificities and their activity within defined chromatin templates are relatively limited. Here we describe purification methods for mammalian TET proteins based on expression in insect cells or in 293T cells. We also briefly summarize a method that can be used to monitor 5-methylcytosine oxidase activity of the purified TET proteins in vitro.

摘要

TET 蛋白介导的 5-甲基胞嘧啶（5mC）氧化途径涉及 5mC 逐步氧化为 5-羟甲基胞嘧啶（5hmC）、5-甲酰胞嘧啶（5fC）和 5-羧基胞嘧啶（5caC）。5fC 和 5caC 可以通过碱基切除修复从 DNA 中去除，该途径的完成导致 5mC 的“去甲基化”，即将修饰碱基转化回胞嘧啶。针对分析 TET 蛋白的 DNA 底物特异性及其在特定染色质模板内活性的体外研究相对有限。在这里，我们描述了基于在昆虫细胞或 293T 细胞中表达的哺乳动物 TET 蛋白的纯化方法。我们还简要总结了一种可用于体外监测纯化 TET 蛋白 5-甲基胞嘧啶氧化酶活性的方法。