Expression of enzymatically active rat liver and human placental catechol-O-methyltransferase in Escherichia coli; purification and partial characterization of the enzyme.
To produce sufficient amounts of recombinant catechol-O-methyltransferase (COMT) for structural and functional studies the coding regions of the rat liver and human placental COMT genes have been introduced into a bacterial expression vector pKEX14. Recombinant COMT was produced in Escherichia coli up to 10% of total bacterial protein after the induction of the T7 RNA polymerase gene with isopropyl-beta-D-thiogalactopyranoside. Both the rat and human enzymes were enzymatically active, soluble and reacted with anti-COMT antiserum in Western blotting. Both enzymes were purified from E. coli cells and partially characterized by determining their specific activity, apparent molecular weight and pI.
Lundström
K
K
Orion Corporation, Laboratory of Molecular Genetics, Helsinki, Finland.
Tilgmann
C
C
Peränen
J
J
Kalkkinen
N
N
Ulmanen
I
I
D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002394:Catechol O-Methyltransferase / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors; D006801:Humans; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D010920:Placenta / Q000201:enzymology*; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification
D011994:Recombinant Proteins; D002394:Catechol O-Methyltransferase