title: Purification and characterization of the recombinant human prostaglandin H synthase-2 expressed in Pichia pastoris.
abstract: Prostaglandin H synthase-1 and -2 (PGHS-1 and PGHS-2, EC 1.14.99.1) are membrane associated glycoproteins that catalyze the first two steps in prostaglandin synthesis. As the enzymes play an important regulatory role in several physiological and pathophysiological processes, recombinant PGHS isoforms are widely used in biomedical research. In the present study, we expressed human PGHS-2 (hPGHS-2) with and without a six histidine sequence tag (His(6) tag) near the amino- or carboxy-terminus of the protein in the Pichia pastoris (P. pastoris) expression system using native or yeast signal sequences. The recombinant His(6) tagged hPGHS-2 was purified using Ni-affinity and anion exchange chromatography, whereas the purification of the C-terminally His(6) tagged hPGHS-2 was more efficient. K(m), k(cat) and IC(50) values were determined to characterize the protein. The data obtained indicate that both the N- and C-terminally His(6) tagged hPGHS-2 are functional and the catalytic properties of the recombinant protein and the enzyme produced in other expression systems are comparable. As the yeast culture is easy to handle, the P. pastoris system could serve as an alternative to the most commonly used baculovirus-insect cell expression system for the production of the recombinant PGHS-2.
authors.affiliation: Department of Chemistry, Tallinn University of Technology, Akadeemia tee 15, 12618 Tallinn, Estonia.
authors.forename: Kaia
authors.initials: K
authors.lastname: Kukk
authors.forename: Reet
authors.initials: R
authors.lastname: Järving
authors.forename: Nigulas
authors.initials: N
authors.lastname: Samel
mesh_terms: D016718:Arachidonic Acid / Q000378:metabolism; D051546:Cyclooxygenase 2 / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D003902:Detergents / Q000737:chemistry; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007213:Indomethacin / Q000737:chemistry; D009842:Oligopeptides / Q000235:genetics / Q000378:metabolism; D010843:Pichia / Q000737:chemistry / Q000201:enzymology / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D013449:Sulfonamides / Q000737:chemistry
chemical_list: D003902:Detergents; C471213:His-His-His-His-His-His; D009842:Oligopeptides; D011994:Recombinant Proteins; D013449:Sulfonamides; D016718:Arachidonic Acid; D006639:Histidine; D051546:Cyclooxygenase 2; C012655:nimesulide; D007213:Indomethacin