Voprosy meditsinskoi khimii (ISSN 0042-8809)

Construction the expression system of mouse inducible nitric oxide synthase in escherichia coli

   
Gervaziev Yu.V., Eldarov M.A., Shkundina I.S., Alexandrova C.C., Voevodskaya M.V., Sokolov N.N.
PubMed Id: 10635536
Year: 1999 vol: 45  issue:5  pages: 416-429
Abstract: In the present work we describe the constructionof 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 vectorpCWori +. As biosynthesis of active iNOS reguires coexpression of calmodulin (CaM), forobtaining functional expression of this protein we conducted amplification of anappropriate site of the library total cDNA a frog Xenopus laevis, then plasmids forcoexpression of calmodulin were constructed under a control tac and T7 promotors.Recombinant iNOS was functionally active as revealed by the analysis of CO-reducedspectrums, detection of derivation NO with the help of reaction conversion HbO2in metHb, and also identification of a molecule NO by EPR method. The output ofrecombinant 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 withthe earlier published results of other investigators. It was established, that theexpressed iNOS is associated to a membrane fraction of cells, thus in the 105 000g-supernatant the activity of an enzyme is not detected. The data on membrane localizationiNOS are inconsistent with general notion this enzyme is soluble.
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Reference: Gervaziev Yu.V., Eldarov M.A., Shkundina I.S., Alexandrova C.C., Voevodskaya M.V., Sokolov N.N., Construction the expression system of mouse inducible nitric oxide synthase in escherichia coli, Voprosy meditsinskoi khimii, 1999, vol: 45(5), 416-429.
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