TY - JOUR
T1 - Characterization of bovine endothelial nitric oxide synthase expressed in E. coli
AU - Martasek, Pavel
AU - Liu, Qing
AU - Liu, Jianwei
AU - Roman, Linda J.
AU - Gross, Steven S.
AU - Sessa, William C.
AU - Masters, Bettie Sue Siler
N1 - Funding Information:
This work was supported, in part, by Grant AQ-1192 from The Robert A. Welch Foundation and National Institutes of Health Grant HL 30050 (to B.S.S.M.), National Institutes of Health Grants HL 50656 and HL 44603 (to S.S.G.), R29-HL 51948 (to W.C.S.), and F32-HL 09224 (to J.L.). The Molecular Cardiobiology Program at Yale is supported by Lederle Pharmaceuticals.
PY - 1996/2/15
Y1 - 1996/2/15
N2 - 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.
AB - 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.
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U2 - 10.1006/bbrc.1996.0238
DO - 10.1006/bbrc.1996.0238
M3 - Article
C2 - 8604992
AN - SCOPUS:0029874753
VL - 219
SP - 359
EP - 365
JO - Biochemical and Biophysical Research Communications
JF - Biochemical and Biophysical Research Communications
SN - 0006-291X
IS - 2
ER -