[Expression constructs of inducible isoform of nitric oxide synthase in Escherichia coli cells].
In the present work we describe the construction of expression system for inducible murine macrophage nitric oxide synthase (iNOS) in E.coli. For this purpose a framework of translation iNOS was cloned in the expression vector pCWori +. As biosynthesis of active iNOS requires coexpression of calmodulin (CaM), for obtaining functional expression of this protein we conducted amplification of an appropriate site of the library total cDNA a frog Xenopus laevis, then plasmids for coexpression of calmodulin were constructed under a control tac and T7 promotors. Recombinant iNOS was functionally active as revealed by the analysis of CO-reduced spectrums, detection of derivation NO with the help of reaction conversion HbO2 in metHb, and also identification of a molecule NO by EPR method. The output of recombinant iNOS at usage of different constructions varied from 10 up to 22 mg/l culture, and specific activity was from 0.42 up to 0.64 U/mg of protein. These data coincide with the earlier published results of other investigators. It was established, that the expressed iNOS is associated to a membrane fraction of cells, thus in the 105,000 g-supernatant the activity of an enzyme is not detected. The data on membrane localization iNOS are inconsistent with general notion this enzyme is soluble.
Gervaziev
Iu V
IuV
Institute of Biomedical Chemistry RAMS, Moscow.
El'darov
M A
MA
Shkundina
I S
IS
Aleksandrova
S S
SS
Voevodskaia
N V
NV
Sokolov
N N
NN
D000818:Animals; D018076:DNA, Complementary; D004926:Escherichia coli / Q000235:genetics*; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics*; D007700:Kinetics; D051379:Mice; D019001:Nitric Oxide Synthase / Q000096:biosynthesis / Q000235:genetics*; D052247:Nitric Oxide Synthase Type II; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D013379:Substrate Specificity; D014981:Xenopus
D018076:DNA, Complementary; D007527:Isoenzymes; D011994:Recombinant Proteins; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse