Cyclooxygenases (COX) are enzymes important in generating the inflammatory response in humans, and are important therapeutic targets for pain regulation. Sequencing tools have identified open reading frames in bacterial, algal, and fungal genomes that encode for proteins predicted to be structurally similar to mammalian (COX) proteins. In this study, putative COX enzymes from Nostoc punctiforme and Rhodobacter sphaeroides were cloned from genomic dna and expressed as SUMO-fusion proteins. Both proteins were expressed in large quantity (&gt; 20 mg/L cell culture) from BL21(DE3) cells. The Nostoc enzyme could be solubilized using buffer containing sodium deoxycholate, and purified using Ni2+ -affinity chromatography. During the affinity purification, the detergent was exchanged either 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), or b-octylglucoside. The Rhodobacter enzyme resisted solubilization with deoxycholate, but could be purified into CHAPS buffer after solubilization with guanidine hydrochloride. For both enzymes, the SUMO fusion partner was removed using SUMO hydrolase, resulting in pure enzyme. The Nostoc enzyme exhibits linoleate dioxygenase activity; activity studies on the Rhodobacter enzyme are ongoing. Additional enzymatic and structural characterizations of the two enzymes are in progress.

Cloning, expression, and characterization of bacterial enzymes with predicted structural similarity to mammalian cyclooxygenases

249th National Meeting (2015)

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