Bacterial Expression of Chimeric Escherichia coli and Trypanosoma brucei DNA Methyltransferases

Our laboratory is interested in DNA and RNA methylation in E. coli and T. brucei as little is known about this form of epigenetic regulation in microorganisms. One methyltransferase being studied at this time is a putative DNA methyltransferase (TbDmt) from Trypanosoma brucei . The exact function of TbDmt is unknown but the protein strongly resembles bacterial DNA methyltransferases such as DNA cytosine methyltransferase (EcDcm) from E. coli . To test our hypothesis that TbDmt is a DNA methyltransferase, we expressed TbDmt in bacteria and created two chimeric protein sequences switching the DNA binding domain and enzymatic domain of EcDcm and TbDmt. Exchanging the DNA binding domain and enzymatic domain of TbDmt with a known methyltransferase may help us discover the function of the enzyme and, if it is a methyltransferase, what DNA sequence is targeted for methylation. Plasmids were made containing the sequences for EcDcm, TbDmt, and both chimeric proteins where the genes are adjacent to the lac operator. E. coli were transformed with the plasmids and expression was induced with IPTG. All four proteins were produced at 20°C, but the proteins with the TbDmt enzymatic domain were less soluble than those with the EcDcm enzymatic domain. The plasmids were re‐isolated after three hours of growth in the presence of IPTG. The plasmids were then digested with eight restriction enzymes blocked by methylation. Each digestion was run on an agarose gel with DNA from uninduced cells and unmethylated phage lambda DNA as controls. EcDcm methylated at its expected site, 5′CCWGG3′, as determined by resistance to PspGI. In addition, there is partial resistance to HpaII in cells containing EcDcm indicating methylation at 5′CCGG3′ or related sequences. The proteins were denatured using guanidium HCl, isolated and analyzed by SDS‐PAGE with Coomassie blue staining. EcDcm and the chimeric protein with the EcDcm enzymatic domain and TbDmt DNA binding domain were successfully purified and were then purified using partial denaturing conditions. These proteins will be tested for methyltransferase activity. TbDMT and the chimera with the TbDMT enzymatic domain were not able to be purified using the guanidium HCl denaturing method so stronger denaturants will be tested in attempt to purify the proteins. Further work will be done in purifying the proteins using both denaturing and nondenaturing conditions to produce functional enzymes for use in methylation assays. This work will contribute to the limited knowledge of methyltransferases in bacteria and protists. Support or Funding Information NIH grant 1R15AI133428‐01 to KTM