High-level expression of mouse inducible nitric oxide synthase in Escherichia coli requires coexpression with calmodulin.
We report a method to generate and purify large quantities of fully active mouse iNOS from E. coli, and show that calmodulin coexpression is essential to generate the active iNOS. E. coli were 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 mumol 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.
Wu
C
C
Department of Immunology, Cleveland Clinic Research Institute, Ohio 44195, USA.
Zhang
J
J
Abu-Soud
H
H
Ghosh
D K
DK
Stuehr
D J
DJ
D000818:Animals; D001483:Base Sequence; D002147:Calmodulin / Q000096:biosynthesis* / Q000302:isolation & purification; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli; D015870:Gene Expression; D006639:Histidine; D006801:Humans; D007527:Isoenzymes / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D019001:Nitric Oxide Synthase / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D016324:Sequence Tagged Sites
D002147:Calmodulin; D017931:DNA Primers; D007527:Isoenzymes; D011994:Recombinant Proteins; D006639:Histidine; D019001:Nitric Oxide Synthase