Purification and characterization of the human recombinant histidine-tagged prostaglandin endoperoxide H synthases-1 and -2.
We have used in vitro mutagenesis to introduce a six residue histidine sequence (His-tag) near the amino terminal end of the human PGHS-1 and -2 and have expressed these proteins using the baculovirus system. The His-tags are located one and two amino acids beyond the signal peptide cleavage sites of PGHS-1 and PGHS-2, respectively, positions that do not affect their activities or sensitivities to nonsteroidal anti-inflammatory drugs. When expressed in sf-21 cells, the His-tagged enzymes have K(m) values for arachidonate, and IC(50) values for inhibition by nonsteroidal anti-inflammatory drugs that are similar to values reported for the nontagged enzymes. The His-tags allowed for purification of the PGHSs by a simplified protocol involving nickel-affinity and anion exchange FPLC chromatography. The specific activities and recoveries for the purified enzymes were as good or better than those reported previously for purification of the non-tagged PGHS. These baculovirus constructs should provide a convenient source for pharmacologic and biophysical studies that require large scale preparation of human PGHSs.
Smith
T
T
Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824, USA.
Leipprandt
J
J
DeWitt
D
D
D000595:Amino Acid Sequence; D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology; D016718:Arachidonic Acid / Q000378:metabolism / Q000494:pharmacology; D016367:Baculoviridae / Q000235:genetics; D001667:Binding, Competitive / Q000187:drug effects; D002460:Cell Line; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; D015870:Gene Expression; D006639:Histidine / Q000235:genetics*; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000494:pharmacology; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009532:Nickel / Q000378:metabolism; D011451:Prostaglandin-Endoperoxide Synthases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000494:pharmacology; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D018411:Spodoptera; D013997:Time Factors
D000894:Anti-Inflammatory Agents, Non-Steroidal; D007527:Isoenzymes; D008565:Membrane Proteins; D011994:Recombinant Proteins; D016718:Arachidonic Acid; D006639:Histidine; D009532:Nickel; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; C496511:PTGS1 protein, human; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases