Detecting S-adenosyl-l-methionine-induced conformational change of a histone methyltransferase using a homogeneous time-resolved fluorescence-based binding assay
Section snippets
G9a protein expression and purification
Human G9a complementary DNA (cDNA) encoding amino acid residues 913 to 1193 was synthesized by GenScript. The fragment was subcloned to pGEX-KG vector using restriction sites EcoRI and XhoI and verified by sequencing. The construct was transformed into Escherichia coli BL21(DE3). A fresh single colony was inoculated into Luria–Bertani (LB) medium and grows to an OD600 of approximately 0.4, and protein expression was induced with 0.5 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) overnight at 16
HTRF assay for measuring binding of BIX-01294 to GST–G9a
BIX-01294 is a known inhibitor of G9a and GLP-1 [13]. Determined crystal structures clearly showed that this compound and its close analogs bind to the peptide–pocket of G9a and/or GLP-1 [24], [25]. To establish an HTRF binding assay between G9a and this compound, we synthesized a biotinylated analog of BIX-01294 called CJP702. CJP702 contains a biotin tag with an ethylene glycol linker attached to the piperidine nitrogen (Fig. 1A). Thus, the tag replaces the benzyl group of BIX-01294. This
Discussion
HTRF- (or TR–FRET)-based assays measuring the binding of an inhibitor probe to its target are commonly used for the identification and characterization of new inhibitors that compete directly with the probe. This method has been successfully demonstrated for protein kinases [21], [22], HSP90 [23], and other targets. The report here is the first demonstration of such an approach being applied to an SET domain-containing HMT. Even though CJP702 used in this study is too specific to be used
Acknowledgments
We thank Martin Klumpp, Zhengtian Yu, Wen Xiong, Jianyong Shou, and Jingquan Dai for their suggestions and help, and we thank Zhui Chen for critical reading of this manuscript.
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2014, TetrahedronCitation Excerpt :Sinefungin (2), a naturally occurring nucleoside isolated from Streptomyces griseolus3 and Streptomyces incarnates,4 has found a prominent place for this purpose. Recent examples of 2 serving in this capacity include analysis of (1) the active site of human histone MTases (lysine MTase, PKMT; and, arginine MTase, PRMT);5–12 (2) the viral mRNA MTase capping process;13–17 (3) methylation of TrmN/Trm14 tRNA in archaea, bacteria, and eukaryotes;18,19 (4) DNA methylation associated with gene expression, particularly related to cancer development;20,21 (5) the phosphoethanolamine MTase in Plasmodium falciparum;22 and, (6) the long range conformational effects of binding in the AdoMet binding domain of histone MTase G9a.23 Sinefungin has also found application beyond MTase investigations.
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These authors contributed equally to this article.