Human endothelial nitric oxide synthase: expression in Escherichia coli, coexpression with calmodulin, and characterization.
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 isoform, but this is the first demonstration that CaM coexpression improves the expression of a constitutive isoform.
Rodríguez-Crespo
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Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco 94143-0446, USA.
Ortiz de Montellano
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D002147:Calmodulin / Q000378:metabolism; D003001:Cloning, Molecular; D004730:Endothelium, Vascular / Q000201:enzymology*; D004926:Escherichia coli; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D019001:Nitric Oxide Synthase / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D013057:Spectrum Analysis
D002147:Calmodulin; D011994:Recombinant Proteins; D019001:Nitric Oxide Synthase