title: Convenient expression, purification and quantitative liquid chromatography-tandem mass spectrometry-based analysis of TET2 5-methylcytosine demethylase.
abstract: 5-Methylcytosine within CpG islands in DNA plays a crucial role in epigenetic transcriptional regulation during metazoan development. Recently, it has been established that the Ten-Eleven Translocation (TET) family, Fe(II)- and 2-oxoglutarate (2OG/αKG)-dependent oxygenases initiate 5-methylcytosine demethylation by iterative oxidation reactions. Mutations in the TET2 gene are frequently detected in patients with myeloid malignancies. Here, we describe the cloning of untagged human TET2 demethylase using Gateway technology and its efficient expression in E. coli. The untagged TET2 enzyme was purified using cation exchange and heparin sepharose chromatography. In addition, a reliable quantitative liquid chromatography-tandem mass spectrometry-based assay was utilized to analyze the activity of TET2 oxygenase. This assay was further used to analyze the activity of a number of clinical TET2 variants with mutations in the 2OG binding sites. Our results demonstrate that the activity of one TET2 mutant, TET2-R1896S, can be restored using an excess of 2OG in the reaction mixture. These studies suggest that dietary 2OG supplements, which are commonly used for several other conditions, may be used to treat some patients with myeloid malignancies harboring TET2-R1896S mutation. Results described in this paper serve as a foundation for better characterization of wild type as well as mutant TET2 demethylases.
authors.affiliation: Division of Pharmaceutical Sciences, School of Pharmacy, University of Missouri-Kansas City, Kansas City, MO 64108, USA.
authors.forename: Mohit
authors.initials: M
authors.lastname: Jaiswal
authors.affiliation: Division of Pharmaceutical Sciences, School of Pharmacy, University of Missouri-Kansas City, Kansas City, MO 64108, USA.
authors.forename: Subhradeep
authors.initials: S
authors.lastname: Bhar
authors.affiliation: Division of Pharmaceutical Sciences, School of Pharmacy, University of Missouri-Kansas City, Kansas City, MO 64108, USA.
authors.forename: Harika
authors.initials: H
authors.lastname: Vemula
authors.affiliation: Division of Pharmaceutical Sciences, School of Pharmacy, University of Missouri-Kansas City, Kansas City, MO 64108, USA.
authors.forename: Swami
authors.initials: S
authors.lastname: Prakash
authors.affiliation: Division of Pharmaceutical Sciences, School of Pharmacy, University of Missouri-Kansas City, Kansas City, MO 64108, USA.
authors.forename: V K Chaithanya
authors.initials: VKC
authors.lastname: Ponnaluri
authors.affiliation: Division of Pharmaceutical Sciences, School of Pharmacy, University of Missouri-Kansas City, Kansas City, MO 64108, USA.
authors.forename: William G
authors.initials: WG
authors.lastname: Gutheil
authors.affiliation: Division of Pharmaceutical Sciences, School of Pharmacy, University of Missouri-Kansas City, Kansas City, MO 64108, USA. Electronic address: mukherjim@umkc.edu.
authors.forename: Mridul
authors.initials: M
authors.lastname: Mukherji
mesh_terms: D002853:Chromatography, Liquid; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D049308:Dioxygenases; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D013058:Mass Spectrometry; D010088:Oxidoreductases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: D004268:DNA-Binding Proteins; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D010088:Oxidoreductases; D049308:Dioxygenases; C541081:TET2 protein, human
keywords: DNA demethylase; Epigenetics; LC-MS/MS; Leukemia; Oxygenase; TET2