Expression and characterization of rat soluble catechol-O-methyltransferase fusion protein.
Rat soluble catechol-O-methyltransferase cDNA was cloned into the pCAL-n-FLAG vector and expressed in Escherichia coli as a fusion protein with a calmodulin-binding peptide tag. The recombinant protein, comprising up to 30% of the total protein in the soluble fraction of E. coli, was purified by calmodulin affinity chromatography and gel filtration. Up to 16 mg of pure recombinant enzyme was recovered per liter of culture. Recombinant catechol-O-methyltransferase, in the bacterial soluble fraction, exhibited the same affinity for adrenaline as rat liver soluble catechol-O-methyltransferase (K(m) 428 [246, 609] microM and 531 [330, 732] microM, respectively), as well as the same affinity for the methyl donor, S-adenosyl-l-methionine (K(m) 27 [9, 45] microM and 38 [21, 55] microM, respectively). In addition, both the recombinant and the liver enzymes displayed the same sensitivity to the inhibitor 3,5-dinitrocatechol (IC(50) 132 [44, 397] nM and 74 [38, 143] nM, respectively), and both had the same catalytic number, respectively, 10.1 +/- 1.5 min(-1) and 8.3 +/- 0.3 min(-1). The purified recombinant enzyme also displayed the same affinity for the substrate as the purified rat liver catechol-O-methyltransferase (K(m) 336 [75, 597] microM and 439 [168, 711] microM, respectively) as well as the same inhibitor sensitivity (IC(50) 44 [19, 101] nM and 61 [33, 111] nM, respectively). This recombinant form of catechol-O-methyltransferase is kinetically identical to the rat liver enzyme. This system provides an easy and quick way of obtaining large amounts of soluble catechol-O-methyltransferase for both pharmacological and structural studies.
Bonifácio
M J
MJ
Department of Research and Development, BIAL, São Mamede do Coronado, 4745-457, Portugal.
Vieira-Coelho
M A
MA
Soares-da-Silva
P
P
D000345:Affinity Labels; D000818:Animals; D002148:Calmodulin-Binding Proteins / Q000096:biosynthesis / Q000235:genetics; D002394:Catechol O-Methyltransferase / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D002846:Chromatography, Affinity; D003001:Cloning, Molecular / Q000379:methods*; D004791:Enzyme Inhibitors / Q000378:metabolism; D004837:Epinephrine / Q000378:metabolism; D004926:Escherichia coli; D020128:Inhibitory Concentration 50; D007700:Kinetics; D010446:Peptide Fragments / Q000096:biosynthesis / Q000235:genetics; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D012995:Solubility
D000345:Affinity Labels; D002148:Calmodulin-Binding Proteins; D004791:Enzyme Inhibitors; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D002394:Catechol O-Methyltransferase; D004837:Epinephrine