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 antiinflammatory drugs, When expressed in sf-21 cells, the His-tagged enzymes have K-m values for arachidonate, and IC50 values for inhibition by nonsteroidal antiinflammatory 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. (C) 2000 Academic Press.