Abstract
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.
Copyright 2000 Academic Press.
Publication types
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Amino Acid Sequence
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Animals
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Anti-Inflammatory Agents, Non-Steroidal / pharmacology
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Arachidonic Acid / metabolism
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Arachidonic Acid / pharmacology
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Baculoviridae / genetics
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Binding, Competitive / drug effects
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Cell Line
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Chromatography, Affinity
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Chromatography, High Pressure Liquid
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Chromatography, Ion Exchange
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Cyclooxygenase 1
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Cyclooxygenase 2
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Gene Expression
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Histidine / genetics*
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Humans
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Isoenzymes / antagonists & inhibitors
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Isoenzymes / chemistry*
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Isoenzymes / genetics
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Isoenzymes / isolation & purification*
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Isoenzymes / pharmacology
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Membrane Proteins
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Molecular Sequence Data
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Molecular Weight
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Nickel / metabolism
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Prostaglandin-Endoperoxide Synthases / chemistry*
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Prostaglandin-Endoperoxide Synthases / genetics
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Prostaglandin-Endoperoxide Synthases / isolation & purification*
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Prostaglandin-Endoperoxide Synthases / pharmacology
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Recombinant Proteins / antagonists & inhibitors
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Recombinant Proteins / chemistry
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Recombinant Proteins / genetics
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Recombinant Proteins / isolation & purification
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Spodoptera
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Time Factors
Substances
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Anti-Inflammatory Agents, Non-Steroidal
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Isoenzymes
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Membrane Proteins
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Recombinant Proteins
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Arachidonic Acid
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Histidine
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Nickel
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Cyclooxygenase 1
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Cyclooxygenase 2
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PTGS1 protein, human
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PTGS2 protein, human
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Prostaglandin-Endoperoxide Synthases