Expression and purification of a human recombinant methyltransferase that repairs damaged proteins.
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 methyl-transferase 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 ovalbumin/mg enzyme/min from S-adenosyl-L-methionine at 37 degrees 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.
MacLaren
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DC
Department of Chemistry and Biochemistry, University of California, Los Angeles 90095-1569, USA.
Clarke
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S
D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000378:metabolism; D001483:Base Sequence; D002848:Chromatography, DEAE-Cellulose; D003001:Cloning, Molecular; D004247:DNA; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids; D026601:Protein D-Aspartate-L-Isoaspartate Methyltransferase; D011496:Protein Methyltransferases / Q000235:genetics* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000235:genetics; D012436:S-Adenosylmethionine / Q000378:metabolism
D011994:Recombinant Proteins; D001224:Aspartic Acid; D012436:S-Adenosylmethionine; D004247:DNA; D011496:Protein Methyltransferases; D026601:Protein D-Aspartate-L-Isoaspartate Methyltransferase