Home
Resources
Publications
Purification and Assay Protocols for Obtaining Highly Active Jumonji C Demethylases

Purification and Assay Protocols for Obtaining Highly Active Jumonji C Demethylases

Swathi Krishnan, Evys Collazo, Patricia A Ortiz-Tello, Raymond C Trievel

Anal Biochem. 2012 Jan 1;420(1):48-53.

PMID: 21925481

Abstract:

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.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
IAR42416060	JMJD2A Active human			Price
IAR42416064	JMJD2D active human			Price

As a specialist in analytical chemical Products, Alfa Chemistry offers consumers a wealth of raw materials and a full range of technologies and services.

QUICK LINKS

HEADQUARTERS

Tel:

Fax:

Email: