Characterization of bovine endothelial nitric oxide synthase expressed in E. coli

Pavel Martasek, Qing Liu, Jianwei Liu, Linda J. Roman, Steven S. Gross, William C. Sessa, Bettie Sue Siler Masters

Research output: Contribution to journalArticlepeer-review

141 Scopus citations


Bovine endothelial constitutive nitric oxide synthase (eNOS) was expressed in E, coli as a soluble, catalytically active enzyme using the pCW expression vector coexpressed with a plasmid, pGroELS, encoding the chaperonins groEL and groES. The E. coli BL21 cultures reproducibly synthesized 6-10 mg of recombinant enzyme per litre of culture. The eNOS protein was purified using 2'5'-ADP Sepharose 4B and appeared as a single band of apparent molecular mass 135 kDa on SDS/PAGE. The recombinant resting enzyme is predominantly high spin with an absorbance maximum at 406 nm. The dithionite-reduced, CO-bound form shows an absorbance maximum at 444 nm. The spectral properties of recombinant eNOS from E. coli are identical to those observed with eNOS from stably transfected HEK 293 cells or from baculovirus expression systems. Enzymatic activity of eNOS from E. coli ranged between 68-135 nmol product formed/min/mg at 25°C, using hemoglobin-NO capture or L-citrulline formation assays. The enzyme is replete with heme and flavins and both activity and [3H]-nitroarginine binding were largely dependent on tetrahydrobiopterin. The heterologous expression of eNOS offers a number of advantages over tissue sources of the protein.

Original languageEnglish (US)
Pages (from-to)359-365
Number of pages7
JournalBiochemical and Biophysical Research Communications
Issue number2
StatePublished - Feb 15 1996

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology


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