Prokaryotic expression, purification and characterization of human cyclooxygenase-2

Abstract

Cyclooxygenase-2 (COX-2) is a key enzyme which catalyzes the conversion of arachidonic acid (AA) into prostaglandins (PGs). It plays an important role in pathophysiological processes, such as tumorigenesis, angiogenesis, inflammation and tumor cell drug resistance. Therefore, COX-2 has been viewed as an important target for cancer therapy. The preparation of COX-2 protein is an important initial step for the subsequent development of COX-2 inhibitors. In this study, we report a strategy to heterologously express truncated human COX-2 (trCOX-2) in Escherichia coli (E. coli) BL21(DE3) host cells. Following denaturation, purification and renaturation, we successfully obtained enzymatically active trCOX-2 containing 257 residues of the C-terminus. Homology modeling and molecular docking analyses revealed that trCOX-2 retained the predicted 3D catalytic domain structure and AA could still bind to its hydrophobic groove. Western blot analysis and ELISA indicated that the trCOX-2 still retained its characteristic antigenicity and binding activity, while COX assays revealed that trCOX-2 maintained its enzyme activity. On the whole, in this study, we provided a novel method to isolate trCOX-2 possessing AA binding and catalytic activities. This study thus lays a foundation to facilitate further investigations of COX-2 and offers a valuable method with which to achieve the prokaryotic expression of a eukaryotic membrane protein.

Gut microbiota-mediated tributyltin-induced metabolic disorder in rats

“…Generally, linoleic acid is reported to prolong and desaturate arachidonic acid, and COX-2 is a key enzyme to catalyze arachidonic acid to produce pro-inammatory factor prostaglandins, which is the rst rate-limiting step in prostaglandin biosynthesis. 39,40 Obviously, the reduction of linoleic acid, 2arachidonylglycerol, prostaglandin E1, and prostaglandin A2 in the TBT group (Fig. 2) suggested that the activation of COX-2 in TBT group was not through the arachidonic acid metabolism.…”

“…COX-2, as a key enzyme located on the membrane of ER, was reported to be N-glycosylated in the ER in the absence of arachidonic acid, and then transported to the Golgi apparatus for modication, before nally being transported back to the ER. 39,41 Furthermore, it has been reported to catalyze the biosynthesis of eicosanoid lipids, affecting the expression of eicosanoid-lysolipids in ER. [42][43][44][45] In addition, PERK is an ER transmembrane protein response to ER stress and could promote apoptosis through activating the downstream proapoptotic transcription factor CHOP.…”

Expression, purification and characterisation of a human anti-CDK4 single-chain variable fragment antibody

“…The negative control for the IPTG-induced E. coli AK2/HB2151 culture supernatant was supernatant obtained without IPTG induction. Due to the absence of AK2 in PBST containing 3% non-fat milk, PBST with 3% non-fat milk served as the negative control for purified AK2, while wells coated with recombinant human prolyl hydroxylase 2 (rhPHD2) [ 27 ], recombinant human cyclooxygenase-2 (rhCOX-2) [ 46 ] or bovine serum albumin (BSA) served as irrelevant protein controls for rhCDK4.…”

Gradient Temperature Raman Spectroscopy of Fatty Acids with One to Six Double Bonds Identifies Specific Carbons and Provides Systematic Three Dimensional Structures

“…While full analysis is beyond the scope of this spectroscopic contribution, we hope that this visualization will promote further understanding of the enzymatics involved. The high degree of specificity of lipoxygenase and cyclooxygenase enzymes [18]- [24] should be reevaluated in light of the fact individual sites on the polyunsaturated fatty acid chain are nonequivalent, and each LC-PUFA molecule has an individual, specific structure incorporating torsion.…”

“…All five of the PUFA we present are metabolized by cyclooxygenase and lipoxygenase enzymes into an extensive suite of oxylipin metabolites [18]- [24].…”