Cloning, Expression, and Purification of a Putative Prostaglandin Synthase from Nostoc as a Sumo Fusion Protein

Abstract

A putative gene for prostaglandin synthase (PGHS) was identified in the genome of the bacteria Nostoc punctiforme. The Nostoc protein is predicted to contain both the heme binding site and cyclooxygenase active site, but lacks sequences attributed to dimer formation and membrane assoiciation. The gene for the putative PGHS was subcloned into an expression vector with a small ubiquitin modifier protein (SUMO) fusion partner and a histidine tag for visualization and isolation. The gene was overexpressed in E. coli BL21 cells, with expression observes both as soluble protein and as insoluble inclusion bodies. The soluble protein was demonstrated to bind heme, as predicted from modeling studies. Purification of the expressed soluble protein was attempted using a Ni affinity resin. Current progress on the purification and characterization of the enzymatic activity of the expressed protein will be reported.