Human endothelial nitric oxide synthase: expression in Escherichia coli, coexpression with calmodulin, and characterization

Archives of Biochemistry and Biophysics
I Rodríguez-Crespo, P R Ortiz de Montellano

Abstract

Human endothelial nitric oxide synthase (eNOS) has been cloned and expressed in Escherichia coli. The spectroscopic properties and specific activity (100-130 nmol x min(-1) x mg(-1) at 37 degrees C) of the recombinant protein are similar to those of the bovine enzyme. FPLC and low-temperature SDS-PAGE indicate that the protein is mostly dimeric in both the absence and presence of tetrahydrobiopterin. Human eNOS thus has a higher tendency to dimerize than the bovine enzyme. A chloramphenicol-resistant, trc promoter-based plasmid has been constructed that allows coexpression of human calmodulin (CaM). Coexpression of CaM increases more than threefold the amount of expressed eNOS, stabilizes the recombinant protein, and significantly augments its specific activity (to 140-170 nmol x min(-1) x mg(-1) at 37 degrees C). The cytochrome c reduction activity is also improved by CaM coexpression. These increases in activity are not achieved by the addition of CaM to eNOS expressed in the absence of CaM. Gel filtration studies suggest that CaM coexpression produces a more elongated eNOS structure and alters the NADPH binding domain. CaM coexpression has been shown previously to be required for successful expression of the inducible NOS is...Continue Reading

Citations

Sep 6, 2012·Biochemistry·Clara Aicart-RamosIgnacio Rodriguez-Crespo
Jul 16, 2008·The Journal of Biological Chemistry·Chun-An ChenJay L Zweier
Oct 19, 2007·DNA and Cell Biology·Monorama TummalaStephen M Black
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