RECOMBINANT HUMAN PHENYLETHANOLAMINE N-METHYLTRANSFERASE - OVERPRODUCTION IN ESCHERICHIA-COLI, PURIFICATION, AND CHARACTERIZATION

The gene encoding phenylethanolamine N-methyltransferase (PNMT) has be en amplified from a human adrenal medulla cDNA library. Following liga tion of the gene into a pET3a-derived expression vector and transforma tion into Escherichia coli BL21(DE3)pLysS, PNMT was expressed, yieldin g about 10% of the soluble protein. The enzyme was purified to homogen eity by ammonium sulfate fractionation followed by ion-exchange chroma tography and gel filtration. The K-m for phenylethanolamine and S-aden osyl-L-methionine were determined to be 130 and 16 mu M, respectively. The enzyme could be inhibited by reagents expected to modify cysteine , arginine, tyrosine, and histidine residues, but not by methyl acetim idate, a reagent expected to modify lysine residues. (C) 1996 Academic Press, Inc.