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.
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Keywords: nitric oxide synthase inducible isoform, nitric oxide, calmodulin, Xenopus Laevis, heterologous expression in E.coli, electron paramagnetic resonance, oxyhemoglbin
Citation:
Gervaziev Yu.V., Eldarov M.A., Shkundina I.S., Alexandrova C.C., Voevodskaya M.V., Sokolov N.N. (1999) Construction the expression system of mouse inducible nitric oxide synthase in Escherichia coli. Voprosy Meditsinskoi Khimii, 45(5), 416-429.
Gervaziev Yu.V. et al. Construction the expression system of mouse inducible nitric oxide synthase in Escherichia coli // Voprosy Meditsinskoi Khimii. - 1999. - V. 45. -N 5. - P. 416-429.
Gervaziev Yu.V. et al., "Construction the expression system of mouse inducible nitric oxide synthase in Escherichia coli." Voprosy Meditsinskoi Khimii 45.5 (1999): 416-429.
Gervaziev, Yu. V., Eldarov, M. A., Shkundina, I. S., Alexandrova, C. C., Voevodskaya, M. V., Sokolov, N. N. (1999). Construction the expression system of mouse inducible nitric oxide synthase in Escherichia coli. Voprosy Meditsinskoi Khimii, 45(5), 416-429.
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