Optimization of baculovirus-mediated expression and purification of hexahistidine-tagged murine DNA (cytosine-C5)-methyltransferase-1 in Spodoptera frugiperda 9 cells.
Enzymatic DNA methylation of carbon 5 of cytosines is an epigenetic modification that plays a role in regulating gene expression, differentiation, and tumorigenesis. DNA (cytosine-C5)-methyltransferase-1 is the enzyme responsible for maintaining established methylation patterns during replication in mammalian cells. It is composed of a large ( approximately 1100 amino acids (a.a.)) amino-terminal region containing many putative regulatory domains and a smaller ( approximately 500 a.a.) carboxy-terminal region containing conserved, catalytic domains. In this study, murine DNA (cytosine C5)-methyltransferase-1, fused to an amino-terminal hexahistidine tag, was expressed by infecting Spodoptera frugiperda cells for 46 h with a recombinant baculovirus carrying the DNA (cytosine-C5)-methyltransferase-1 cDNA. A total of 3 x 10(8) infected S. frugiperda cells yielded approximately 1 mg of full-length, hexahistidine-tagged DNA (cytosine-C5)-methyltransferase-1, which was purified approximately 450-fold from RNase-treated S. frugiperda cell extracts by nickel affinity chromatography. The characterization of hexahistidine-tagged DNA (cytosine-C5)-methyltransferase-1 through DNA methylation and inhibitor-binding assays indicated that the purified enzyme had at least a 30-fold higher catalytic efficiency with hemimethylated double-stranded oligodeoxyribonucleotide substrates than unmethylated substrates and was most active with small oligodeoxyribonucleotide substrates with a capacity for forming stem-loop structures. The expression and purification procedures reported here differ significantly from the original reports of baculovirus-mediated hexahistidine-tagged DNA (cytosine-C5)-methyltransferase-1 expression and purification by nickel affinity chromatography and provide a consistent yield of active enzyme.
Brank
Adam S
AS
Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, Nebraska 68198, USA.
Van Bemmel
Dana M
DM
Christman
Judith K
JK
D000818:Animals; D016367:Baculoviridae / Q000378:metabolism*; D001665:Binding Sites; D015153:Blotting, Western; D002460:Cell Line; D002846:Chromatography, Affinity; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000096:biosynthesis* / Q000302:isolation & purification*; D019175:DNA Methylation; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005437:Flucytosine / Q000378:metabolism; D006639:Histidine / Q000378:metabolism*; D007313:Insecta; D007700:Kinetics; D051379:Mice; D009841:Oligonucleotides / Q000378:metabolism; D018411:Spodoptera; D013997:Time Factors
D018076:DNA, Complementary; D009841:Oligonucleotides; D006639:Histidine; D005437:Flucytosine; D004248:DNA (Cytosine-5-)-Methyltransferases