High-level expression of functional rat neuronal nitric oxide synthase in Escherichia coli

Linda J. Roman, Essam A. Sheta, Pavel Martasek, Steven S. Gross, Qing Liu, Bettie Sue Siler Masters

Research output: Contribution to journalArticlepeer-review

241 Scopus citations

Abstract

The neuronal nitric oxide synthase (nNOS) has been successfully overexpressed in Escherichia coli, with average yields of 125-150 nmol (20- 24 mg) of enzyme per liter of cells. The cDNA for nNOS was subcloned into the pCW vector under the control of the tac promotor and was coexpressed with the chaperonins groEL and groES in the protease-deficient BL21 strain of E. coli. The enzyme produced is replete with heme and flavins and, after overnight incubation with tetrahydrobiopterin, contains 0.7 pmol of tetrahydrobiopterin per pmol of nNOS. nNOS is isolated as a predominantly high-spin heme protein and demonstrates spectral properties that are identical to those of nNOS isolated from stably transfected human kidney 293 cells. It binds N(ω)- nitroarginine dependent on the presence of bound tetrahydrobiopterin and exhibits a K(d) of 45 nM. The enzyme is completely functional; the specific activity is 450 nmol/min per mg. This overexpression system will be extremely useful for rapid, inexpensive preparation of large amounts of active nNOS for use in mechanistic and structure/function studies, as well as for drug design and development.

Original languageEnglish (US)
Pages (from-to)8428-8432
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume92
Issue number18
DOIs
StatePublished - Aug 29 1995

ASJC Scopus subject areas

  • General

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