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Bacterial Expression of Chimeric Escherichia coli and Trypanosoma brucei DNA Methyltransferases
Author(s) -
Taber Cassandra Carol,
Militello Kevin
Publication year - 2019
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2019.33.1_supplement.621.13
Subject(s) - biology , methyltransferase , dna methyltransferase , dna methylation , microbiology and biotechnology , plasmid , dna , methylation , biochemistry , gene , gene expression
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 T5 promoter and lac operator. E. coli lacking a cytosine DNA methylation pathway 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 DNA binding domain were less soluble than the other two. 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 an uninduced cell 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. We are currently testing restriction enzyme sensitivity in the samples containing full length TbDmt and chimeric TbDMTs. Further work will be done to improve the conditions in which the chimeric proteins are produced to enhance folding of the potential DNA methyltransferases. In summary, this work contributes to our limited knowledge of DNA methylation enzymes and targets in bacteria and protists. Support or Funding Information NIH, 1R15AI133428‐01 This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .