Recombinant human phenylethanolamine N-methyltransferase: overproduction in Escherichia coli, purification, and characterization.
The gene encoding phenylethanolamine N-methyltransferase (PNMT) has been amplified from a human adrenal medulla cDNA library. Following ligation of the gene into a pET3a-derived expression vector and transformation into Escherichia coli BL21(DE3)pLysS, PNMT was expressed, yielding about 10% of the soluble protein. The enzyme was purified to homogeneity by ammonium sulfate fractionation followed by ion-exchange chromatography and gel filtration. The Km for phenylethanolamine and S-adenosyl-L-methionine were determined to be 130 and 16 microM, respectively. The enzyme could be inhibited by reagents expected to modify cysteine, arginine, tyrosine, and histidine residues, but not by methyl acetimidate, a reagent expected to modify lysine residues.
Caine
J M
JM
Victorian College of Pharmacy, Monash University, 381 Royal Parade, Parkville, Victoria, 3052, Australia.
Macreadie
I G
IG
Grunewald
G L
GL
McLeish
M J
MJ
D000595:Amino Acid Sequence; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D017931:DNA Primers; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression / Q000235:genetics; D006801:Humans; D007096:Imidoesters / Q000494:pharmacology; D007460:Iodoacetamide / Q000494:pharmacology; D007700:Kinetics; D008741:Methyl Methanesulfonate / Q000031:analogs & derivatives / Q000494:pharmacology; D008969:Molecular Sequence Data; D010625:Phenylethanolamine N-Methyltransferase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D010658:Phenylglyoxal / Q000494:pharmacology; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics* / Q000302:isolation & purification; D012436:S-Adenosylmethionine / Q000378:metabolism; D013774:Tetranitromethane / Q000494:pharmacology
D017931:DNA Primers; D007096:Imidoesters; D011994:Recombinant Proteins; C014674:methyl methanethiosulfonate; D012436:S-Adenosylmethionine; D008741:Methyl Methanesulfonate; C010222:methyl acetimidate; D010625:Phenylethanolamine N-Methyltransferase; D013774:Tetranitromethane; D004047:Diethyl Pyrocarbonate; D010658:Phenylglyoxal; D007460:Iodoacetamide