Expression and characterization of rat soluble catechol-O-methyltransferase fusion protein

Rat soluble catechol-O-methyltransferase cDNA was cloned into the pCAL-n-FL AG vector and expressed in Escherichia coli as a fusion protein with a calm odulin-binding peptide tag. The recombinant protein, comprising up to 30% o f the total protein in the soluble fraction of E. coli, was purified by cal modulin affinity chromatography and gel filtration. Up to 16 mg of pure rec ombinant enzyme was recovered per liter of culture. Recombinant catechol-O- methyltransferase, in the bac. terial soluble fraction, exhibited the same affinity for adrenaline as rat liver soluble catechol-O-methyltransferase ( K-m 428 [246, 609] muM and 531 [330, 732] muM, respectively), as well as th e same affinity for the methyl donor, S-adenosyl-L-methionine (K-m 27 [9, 4 5] muM and 38 [21,55] muM, respectively). In addition, both the recombinant and the liver enzymes displayed the same sensitivity to the inhibitor 3,5- dinitrocatechol (IC50 132 [44, 397] nM and 74 [38,143] nM, respectively), a nd both had the same catalytic number, respectively, 10.1 +/- 1.5 min(-1) a nd 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-methyl transferase (K-m 336 [75, 597] muM and 439 [168, 711] muM, respectively) as well as the same inhibitor sensitivity (IC50 44 [19, 101] nM and 61 [33, 1 11] nM, respectively). This recombinant form of catechol-O-methyltransferas e is kinetically identical to the rat liver enzyme. This system provides an easy and quick way of obtaining large amounts of soluble catechol-O-methyl transferase for both pharmacological and structural studies. (C) 2001 Acade mic Press.