Preparation And Characterization Of TET Mutants

TET (Ten-Eleven-Translocation) proteins are Fe2+and ketoglutaric acid-dependent Dioxygenase, which can catalyze the conversion of5-methylcytosine(5mC) to5-hydroxymethylcytosine (5hmC). By regulating the relative contentof5mC and5hmC in DNA, TET proteins play important roles in the regulationof gene expression, genetic imprinting and tumor formation.TET proteins family includes three members: TET1, TET2and TET3. Thesequences of human TET1, TET2, TET3and zebrafish TET2were cloned intothe pET28b vector. After codon optimization by site-directed mutagenesis,16TET mutants with different length were prepared. It was found thatoverexpression of TET mutants in E.coli leaded to the formation of inclusionbody, which could be partially alleviated by low culture temperature, fusionwith SUMO-tag, coexperssion with trigger factor or supplementation withchemical chaperone in medium. On the other hand, we investigated the refoldingcondition of denatured TET mutants, and developed the preparation methods ofTET mutants from inclusion body. By circular dichroism and fluorescencetechniques, we found the refolded TET proteins had native-like secondary andtertiary structure.Our results not only contribute to the development of high-efficientpreparation method of recombinant TET proteins, but also are helpful for us toelucidate the folding mechanism of TET proteins.