Overproduction, purification and characterization of M.EcoHK31I, a bacterial methyltransferase with two polypeptides

Abstract

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 α and β 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 3.0×105 units per mg of protein. The respective denatured molecular masses of polypeptides α and β 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 μM 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.