Large-scale expression, purification, and characterization of an engineered prostacyclin-synthesizing enzyme with therapeutic potential.
Recently, we reported that a novel hybrid enzyme (TriCat enzyme), engineered by linking human cyclooxygenase-2 (COX-2) with prostacyclin (PGI(2)) synthase (PGIS) together through a transmembrane domain, was able to directly integrate the triple catalytic (TripCat) functions of COX-2 and PGIS and effectively convert arachidonic acid (AA) into the vascular protector, PGI(2) [K.H. Ruan, H. Deng, S.P. So, Biochemistry 45 (2006) 14003-14011]. In order to confirm the important biological activity and evaluate its therapeutic potential, it is critical to characterize the properties of the enzyme using the purified protein. The TriCat enzyme cDNA was subcloned into a baculovirus vector and its protein was expressed in Sf-9 cells in large-scale with a high-yield ( approximately 4% of the total membrane protein), as confirmed by Western blot and protein staining. The Sf-9 cells' membrane fraction, rich in TriCat enzyme, exhibited strong TriCat functions (K(m)=3 microM and K(cat)=100 molecules/min) for the TriCat enzyme and was 3-folds faster in converting AA to PGI(2) than the combination of the individual COX-2 and PGIS. Another superiority of the TriCat enzyme is its dual effect on platelet aggregation: it completely inhibited platelet aggregation at the low concentration of 2 microg/ml and then displayed the ability to reverse the initially aggregated platelets to their non-aggregated state. Furthermore, multiple substrate-binding sites were confirmed in the single protein by high-resolution NMR spectroscopy, using partially purified TriCat enzyme. These studies have clearly demonstrated that the isolated TriCat enzyme protein functions in the selective biosynthesis of the vascular protector, PGI(2), and revealed its potential for anti-thrombosis therapeutics.
Ruan
Ke-He
KH
Department of Pharmacological and Pharmaceutical Sciences, Center for Experimental Therapeutics and PharmacoInformatics, College of Pharmacy, University of Houston, Science & Research Building 2, Room 521, Houston, TX 77204-5037, USA. khruan@uh.edu
So
Shui-Ping
SP
Wu
Hanjing
H
Cervantes
Vanessa
V
D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D016367:Baculoviridae / Q000235:genetics; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D051546:Cyclooxygenase 2 / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D018076:DNA, Complementary / Q000235:genetics; D015195:Drug Design; D011464:Epoprostenol / Q000096:biosynthesis; D005822:Genetic Vectors; D006801:Humans; D066298:In Vitro Techniques; D019746:Intramolecular Oxidoreductases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D007700:Kinetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D010974:Platelet Aggregation / Q000187:drug effects; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D018411:Spodoptera
D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D016718:Arachidonic Acid; D003577:Cytochrome P-450 Enzyme System; D011464:Epoprostenol; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D019746:Intramolecular Oxidoreductases; C022461:prostacyclin synthetase