[Expression constructs of inducible isoform of nitric oxide synthase in Escherichia coli cells]

Vopr Med Khim. 1999 Sep-Oct;45(5):416-29.
[Article in Russian]

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 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.

MeSH terms

  • Animals
  • DNA, Complementary
  • Escherichia coli / genetics*
  • Isoenzymes / biosynthesis
  • Isoenzymes / genetics*
  • Kinetics
  • Mice
  • Nitric Oxide Synthase / biosynthesis
  • Nitric Oxide Synthase / genetics*
  • Nitric Oxide Synthase Type II
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / genetics
  • Substrate Specificity
  • Xenopus

Substances

  • DNA, Complementary
  • Isoenzymes
  • Recombinant Proteins
  • Nitric Oxide Synthase
  • Nitric Oxide Synthase Type II
  • Nos2 protein, mouse