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Expression, Purification, and Characterization of a Putative Cyclooxygenase from Nostoc punctiforme
Author(s) -
Tang Huasong,
Backus Kevin,
Selinsky Barry S
Publication year - 2011
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.25.1_supplement.923.5
Subject(s) - fusion protein , nostoc , biochemistry , open reading frame , gene , plasmid , biology , affinity chromatography , enzyme , genome , ubiquitin , microbiology and biotechnology , peptide sequence , genetics , recombinant dna , cyanobacteria , bacteria
The use of sequence‐dependent profile searches of bacterial and fungal genomes have uncovered the existence of open reading frames encoding proteins predicted to be similar to mammalian cyclooxygenase (COX) proteins. In this study, a putative COX from Nostoc punctiforme was identified, expressed, purified, and characterized. The gene (Npun_R5469) was cloned into an expression plasmid with an N‐terminal small ubiquitin related modifier (SUMO) fusion partner, and overexpressed in BL21(DE3) cells. The largely soluble fusion protein was purified by Ni2+‐affinity chromatography, cleaved with SUMO‐hydrolase, and further purified by removal of the SUMO fusion partner. Kinetic characterization of the Nostoc protein product is in progress, and should provide insight into the evolutionary origin of mammalian COX enzymes.