Abstract
A method is described to express and purify human DNA (cytosine-5) methyltransferase (human DNMT1) using a protein splicing (intein) fusion partner in a baculovirus expression vector. The system produces approximately 1 mg of intact recombinant enzyme >95% pure per 1.5 x 10(9) insect cells. The protein lacks any affinity tag and is identical to the native enzyme except for the two C-terminal amino acids, proline and glycine, that were substituted for lysine and aspartic acid for optimal cleavage from the intein affinity tag. Human DNMT1 was used for steady-state kinetic analysis with poly(dI-dC).poly(dI-dC) and unmethylated and hemimethylated 36- and 75-mer oligonucleotides. The turnover number (k(cat)) was 131-237 h(-1) on poly(dI-dC).poly(dI-dC), 1.2-2.3 h(-1) on unmethylated DNA, and 8.3-49 h(-1) on hemimethylated DNA. The Michaelis constants for DNA (K(m)(CG)) and S-adenosyl-L-methionine (AdoMet) (K(m)(AdoMet)) ranged from 0.33-1.32 and 2.6-7.2 microM, respectively, whereas the ratio of k(cat)/K(m)(CG) ranged from 3.9 to 44 (237-336 for poly(dI-dC).poly(dI-dC)) x 10(6) M(-1) h(-1). The preference of the enzyme for hemimethylated, over unmethylated, DNA was 7-21-fold. The values of k(cat) on hemimethylated DNAs showed a 2-3-fold difference, depending upon which strand was pre-methylated. Furthermore, human DNMT1 formed covalent complexes with substrates containing 5-fluoro-CNG, indicating that substrate specificity extended beyond the canonical CG dinucleotide. These results show that, in addition to maintenance methylation, human DNMT1 may also carry out de novo and non-CG methyltransferase activities in vivo.
Highlights
A method is described to express and purify human DNA methyltransferase using a protein splicing fusion partner in a baculovirus expression vector
Expression and Purification of the Biologically Active Human DNA (Cytosine-5) Methyltransferase—Co-transfection of the human DNMT1 transfer vector, pVICHMT, with linear Autographa californica nuclear polyhedrosis virus (AcNPV) DNA (BaculoGold DNA, PharMingen) resulted in homologous recombination of the polyhedrin promoter and the DNMT1 cDNA carried by the construct (Fig. 1, A and B)
Several clones had double homologous integration with the human DNMT1 coding sequence lying downstream of the polyhedrin promoter, and one, VICHMT8, was used for all further expression and purification studies
Summary
Human DNA (Cytosine-5) Methyltransferase Transfer Vector—Human DNMT1 expression constructs were derived from pBKSHMT5.0 (b) Recombinant DNA (Cytosine-5) Methyltransferase Purification—For protein purification, infected cells (5.6 ϫ 108) were resuspended in 15 ml of buffer M (50 mM Tris-HCl, pH 7.4, 1 mM Na2EDTA, protease inhibitor mixture containing 4-(2-aminoethyl)benzenesulfonyl fluoride, pepstatin A, E64, bestatin, leupeptin and aprotinin (Sigma), 0.2% (v/v) per ml of cell extract, 7 g/ml phenylmethylsulfonyl fluoride, and 500 mM NaCl). Purification from larger numbers (Ͼ1 ϫ 109) of cells required an initial 40 –70% ammonium sulfate fractionation This supernatant was loaded on a chitin bead column (New England Biolabs) equilibrated with buffer M at 0.4 ml/min. A typical reaction contained S-adenosyl-L- [methyl-3H]methionine (AdoMet) (specific activity 15 Ci/ mmol, Amersham Pharmacia Biotech), substrate DNA, and enzyme in assay buffer (50 mM Tris-HCl, pH 7.8, 1 mM Na2EDTA, pH 8.0, 1 mM DTT, 7 g/ml phenylmethylsulfonyl fluoride, 5% glycerol, and 100 g/ml bovine serum albumin). The full-length duplexes were purified on a 15% polyacrylamide gel, dissolved in TE, and stored at Ϫ20 °C
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