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Titolo:
Expression and characterization of rat soluble catechol-O-methyltransferase fusion protein
Autore:
Bonifacio, MJ; Vieira-Coelho, MA; Soares-da-Silva, P;
Indirizzi:
BIAL, Dept Res & Dev, P-4745457 Sao Mamede Coronado, Portugal BIAL Sao Mamede Coronado Portugal P-4745457 ao Mamede Coronado, Portugal
Titolo Testata:
PROTEIN EXPRESSION AND PURIFICATION
fascicolo: 1, volume: 23, anno: 2001,
pagine: 106 - 112
SICI:
1046-5928(200110)23:1<106:EACORS>2.0.ZU;2-1
Fonte:
ISI
Lingua:
ENG
Soggetto:
TIGHT-BINDING INHIBITORS; PARKINSONIAN-PATIENTS; KINETICS; BRAIN; LIVER; PURIFICATION; TOLCAPONE; METHYLATION; DISEASE; ENZYME;
Keywords:
recombinant catechol-O-methyltransferase; fusion protein; kinetic analysis; COMT inhibitor;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
32
Recensione:
Indirizzi per estratti:
Indirizzo: Soares-da-Silva, P BIAL, Dept Res & Dev, P-4745457 Sao Mamede Coronado, Portugal BIAL Sao Mamede Coronado Portugal P-4745457 Portugal
Citazione:
M.J. Bonifacio et al., "Expression and characterization of rat soluble catechol-O-methyltransferase fusion protein", PROT EX PUR, 23(1), 2001, pp. 106-112

Abstract

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 sameaffinity 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 (IC50 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 thesame 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 (IC50 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.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 14/07/20 alle ore 19:52:31