Russian Journal

ISSN 0042-8809

Issue:
Volume 45, issue 5

Title: CONSTRUCTION THE EXPRESSION SYSTEM OF MOUSE INDUCIBLE NITRIC OXIDE SYNTHASE IN ESCHERICHIA COLI.

Authors: Yu.V.GERVAZIEV*, M.A.ELDAROV**, I.S.SHKUNDINA*, A.A.ALEXANDROVA*, M.V.VOEVODSKAYA***, N.N.SOKOLOV*.

Address:
*Institute of Biomedical Chemistry RAMS, Pogodinskaya str. 10, 119832 Moscow; fax: (095) 245-0857; e-mail: yura@medic.ibmh.msk.su

***Institute of Physical Chemistry, RAS

Abstract:

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 reguires 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.

Key words: nitric oxide synthase inducible isoform, nitric oxide, calmodulin, Xenopus Laevis, heterologous expression in E.coli, electron paramagnetic resonance, oxyhemoglbin.

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