Characterization of bovine endothelial nitric oxide synthase expressed in E. coli.
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 liter 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 degrees 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.
Martasek
P
P
Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284-7760, USA.
Liu
Q
Q
Liu
J
J
Roman
L J
LJ
Gross
S S
SS
Sessa
W C
WC
Masters
B S
BS
D000818:Animals; D001483:Base Sequence; D002245:Carbon Dioxide / Q000378:metabolism; D002417:Cattle; D003001:Cloning, Molecular; D004730:Endothelium, Vascular / Q000201:enzymology*; D004926:Escherichia coli; D007700:Kinetics; D008969:Molecular Sequence Data; D019001:Nitric Oxide Synthase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D013053:Spectrophotometry
D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D002245:Carbon Dioxide; D019001:Nitric Oxide Synthase