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 depe ndent upon the isolation and characterization of the cellular componen ts of the DNA methylation system. The enzyme which methylates DNA in e ukaryotic cells is a C-5 cytosine DNA methyltransferase. Historically, the characterization of this enzyme has been limited by its availabil ity 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 co-pur ified with inhibitory RNA which was removed by treatment with ribonucl ease A. Like its non-recombinant counterpart, the recombinant enzyme i s activated by hemi-methylation. A direct steady-state kinetic compari son between the recombinant baculovirus-expressed enzyme with its MEL cell-derived counterpart is presented. (C) 1997 Academic Press