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

• Type: Article in International Scientific Journal
• Date: 2001

Authors

bonifacio, mj (Author) Other; vieira-coelho, ma (Author) FMUP; soares-da-silva, p (Author) FMUP

Journal

Title: Protein Expression and Purification

Vol. 23

Pages: 106-112

ISSN: 1046-5928

Publisher: Elsevier

Indexing

ISI Web of Knowledge - 15 Citations

Scopus - 15 Citations

Scientific classification

FOS: Natural sciences > Biological sciences

Other information

Authenticus ID: P-000-T7H

DOI: 10.1006/prep.2001.1477

Abstract (EN): 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 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 the same affinity for the methyl donor, S-adenosyl-L-methionine (K(m) 27 [9, 45] 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 (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] muM and 439 [168, 711] muM, 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. (C) 2001 Academic Press.

Language: English

Type (Professor's evaluation): Scientific

No. of pages: 7