Purification of recombinant human COX-1 and COX-2.
Human recombinant COX-1 and COX-2 was prepared in a purified form. The genes were cloned and expressed in insect cells, extracted by detergents, and purified by ion-exchange followed by size exclusion chromatography. Insect cells from 10 L fermentation yielded 2.3 mg of COX-1 with an overall yield of 75%, and 29 mg of COX-2 with an overall yield of 45%. Enzyme prepared in this manner was fully active and proved to be useful in biophysical studies including direct binding studies. The COX-2 provided material that was subsequently used in X-ray crystallography studies.
Gierse
James K
JK
Pfizer Global Research and Development, Inflammation Research, Chesterfield, MO, USA. James.k.gierse@pfizer.com
D000818:Animals; D002850:Chromatography, Gel / Q000379:methods*; D002852:Chromatography, Ion Exchange / Q000379:methods*; D003001:Cloning, Molecular; D051545:Cyclooxygenase 1 / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D051546:Cyclooxygenase 2 / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007313:Insecta / Q000166:cytology; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D011994:Recombinant Proteins; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2