Purification and characterization of recombinant baculovirus-expressed mouse DNA methyltransferase.
DNA methylation is essential for normal embryonic development in mice. An understanding of how DNA methylation is controlled is largely dependent upon the isolation and characterization of the cellular components of the DNA methylation system. The enzyme which methylates DNA in eukaryotic cells is a C-5 cytosine DNA methyltransferase. Historically, the characterization of this enzyme has been limited by its availability and purity. Here, we present a single-step purification of 4 mg of baculovirus-expressed mouse DNA methyltransferase containing a nickel-affinity leader peptide. The recombinant DNA methyltransferase copurified with inhibitory RNA which was removed by treatment with ribonuclease A. Like its non-recombinant counterpart, the recombinant enzyme is activated by hemi-methylation. A direct steady-state kinetic comparison between the recombinant baculovirus-expressed enzyme with its MEL cell-derived counterpart is presented.
Glickman
J F
JF
Department of Chemistry and Program in Biochemistry and Molecular Biology, University of California, Santa Barbara 93106, USA.
Flynn
J
J
Reich
N O
NO
D000818:Animals; D016367:Baculoviridae; D001483:Base Sequence; D002460:Cell Line; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D015254:DNA Modification Methylases / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism*; D007700:Kinetics; D004915:Leukemia, Erythroblastic, Acute; D051379:Mice; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D018411:Spodoptera; D013379:Substrate Specificity; D014162:Transfection; D014407:Tumor Cells, Cultured
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D015254:DNA Modification Methylases