Overproduction, purification and characterization of M.EcoHK31I, a bacterial methyltransferase with two polypeptides.
The two overlapping genes coding for EcoHK31I methyltransferase have previously been cloned, sequenced and expressed [Lee, Kam and Shaw (1995) Nucleic Acids Res. 23, 103-108]. Here we describe protocols developed to purify polypeptides alpha and beta together or separately, to apparent homogeneity by various chromatographic media. M.EcoHK31I is a heterodimer with a native molecular mass of 61 kDa. Its specific activity towards non-methylated lambda DNA was 3.0 x 10(5) units per mg of protein. The respective denatured molecular masses of polypeptides alpha and beta were 38 and 23 kDa, and their pI values were 8.7 and 6.8. Initial rate kinetic parameters of the native enzyme were 2.0 nM, 0.58 microM and 3 min-1 for KmDNA, KmAdoMet and kcat. respectively, where AdoMet stands for S-adenosyl-L-methionine. Fully active enzyme was reconstituted by co-purifying the two separately synthesized polypeptides, and activity assays confirmed our previous finding that two polypeptides were needed to methylate substrate DNA.
Lee
K F
KF
Department of Biochemistry, The Chinese University of Hong Kong.
Liaw
Y C
YC
Shaw
P C
PC
D000595:Amino Acid Sequence; D004247:DNA / Q000378:metabolism; D015257:DNA-Cytosine Methylases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D005807:Genes, Overlapping; D007700:Kinetics; D008745:Methylation; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012436:S-Adenosylmethionine / Q000378:metabolism
D010455:Peptides; D011994:Recombinant Proteins; D012436:S-Adenosylmethionine; D004247:DNA; C092246:DNA modification methylase EcoHK31I; D015257:DNA-Cytosine Methylases