Purification and assay protocols for obtaining highly active Jumonji C demethylases
Purification and assay protocols for obtaining highly active Jumonji C demethylases
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DOI：
10.1016/j.ab.2011.08.034
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发表时间：
2012-01-01
影响因子：
2.9
通讯作者：
Trievel, Raymond C.
中科院分区：
文献类型：
--
作者：
Krishnan, Swathi;Collazo, Evys;Trievel, Raymond C.
Jumonji C（JmjC）赖氨酸脱甲基酶（KDM）是Fe（II）依赖性羟化酶，催化组蛋白和非组蛋白中甲基赖氨酸残基的氧化脱甲基。这些酶在调节细胞过程如基因表达、细胞周期进程和干细胞自我更新和分化中起重要作用。尽管它们具有生物学重要性，但重组形式的JmjC KDM通常显示出低酶活性，并且仍然难以以高活性形式分离。在这里，我们提出了一个简单的亲和纯化方案链球菌（II）标记的JmjC KDM，最大限度地减少过渡态金属离子的污染，产生高活性和纯酶。我们还描述了一个优化的连续荧光检测KDM的检测甲醛生产过程中，通过使用甲醛脱氢酶的偶联反应。使用这些方法对人KDM JMJD2A和JMJD2D进行纯化和动力学分析，得到的活性显著高于先前报道的这些酶的活性，这些酶与黄素依赖性KDM LSD 1的活性相当。此外，我们表明，JMJD2A表现出较低的催化效率对组蛋白肽轴承化学安装的三甲基赖氨酸类似物相比，真正的三甲基化基板。这里描述的方法广泛适用于其他JmjC KDM，促进其生化表征和高通量筛选应用。(C)2011 Elsevier Inc. All rights reserved.
Jumonji C (JmjC) lysine demethylases (KDMs) are Fe(II)-dependent hydroxylases that catalyze the oxidative demethylation of methyllysine residues in histones and nonhistone proteins. These enzymes play vital roles in regulating cellular processes such as gene expression, cell cycle progression, and stem cell self-renewal and differentiation. Despite their biological importance, recombinant forms of JmjC KDMs generally display low enzymatic activity and have remained challenging to isolate in a highly active form. Here we present a simple affinity purification scheme for Strep(II)-tagged JmjC KDMs that minimizes contamination by transition state metal ions, yielding highly active and pure enzyme. We also describe an optimized continuous fluorescent assay for KDMs that detects formaldehyde production during demethylation via a coupled reaction using formaldehyde dehydrogenase. Purification and kinetic analysis of the human KDMs JMJD2A and JMJD2D using these methods yielded activities substantially higher than those previously reported for these enzymes, which are comparable to that of the flavin-dependent KDM LSD1. In addition, we show that JMJD2A exhibited a lower catalytic efficiency toward a histone peptide bearing a chemically installed trimethyllysine analog compared with a bona fide trimethylated substrate. The methodology described here is broadly applicable to other JmjC KDMs, facilitating their biochemical characterization and high-throughput screening applications. (C) 2011 Elsevier Inc. All rights reserved.