We report the construction of a plasmid (pDM2x) containing the coding sequence of the more acidic isozyme II of the human protein-L-isoaspartate (D-aspartate) O-methyltransferase (EC 2.1.1.77) and the overexpression and purification of the recombinant protein. This intracellular enzyme is present in all tissues and can catalyze the first step of a repair reaction where proteins containing abnormal L-isoaspartyl (or D-aspartyl) residues can be converted to forms containing normal L-aspartyl residues. When the methyltransferase cDNA is expressed in Escherichia coli strain BL21 (DE3) under the T7 phage promoter, we find that active enzyme is produced in amounts up to 20% of the total soluble protein. We have developed a rapid and efficient purification method utilizing a one column-step nonaffinity fractionation that allows for the preparation of 10.2 mg of homogeneous enzyme from 2.6 liters of Luria-Bertani broth culture in less than 24 h. The product is soluble and fully active (10,000 pmol of methyl groups transferred to oval-bumin/mg enzyme/min from S-adenosyl-L-methionine at 37 o C). Conditions have been developed to concentrate this enzyme to 30 mg/ml. Analyses of the purified enzyme by N-terminal Edman sequencing and electrospray mass spectroscopy reveal that it is identical to the human isozyme II with the exception that the N-terminal alanine residue is not acetylated.