High-Level Expression of Mouse Inducible Nitric Oxide Synthase inEscherichia coliRequires Coexpression with Calmodulin

Abstract

We report a method to generate and purify large quantities of fully active mouse iNOS fromE. coli,and show that calmodulin coexpression is essential to generate the active iNOS.E. coliwere transformed with a plasmid containing mouse iNOS with a six-histidine tag on its N-terminus or were cotransformed with piNOS and a distinct plasmid that contained human calmodulin. Protein expression was induced by IPTG followed by culture at room temperature. Coexpression with calmodulin enabled production of active iNOS (20 mg/L culture), of which half could be recovered in pure form by sequential metal chelate and 2′, 5′ ADP Sepharose chromatography. The calmodulin-replete iNOS was dimeric, contained normal quantities of heme, flavins, and tightly bound calmodulin, and had high NO synthesis activity (0.7–1.2 μmol NO/min per mg). In contrast, calmodulin-deficient iNOS was monomeric, devoid of flavins and heme, and had no NO synthesis activity. We conclude that calmodulin is essential to fold and stabilize mouse iNOS.