Cloning and expression of a rat neuronal nitric oxide synthase coding sequence in a baculovirus/insect cell system.
A DNA sequence encoding rat neuronal NO synthase (nNOS) was isolated and cloned into the baculovirus expression vector pVL1393 to generate pVLRBNOS. Transfection of Spodoptera frugiperda Sf-21 cells with the construct pVLRBNOS resulted in the synthesis of high levels of neuronal NO synthase. Analysis of the expression pattern revealed soluble, enzymatically active NO synthase in the cytoplasm of cell extracts. Active enzyme could also be purified from culture supernatants using 2'-5' ADP sepharose affinity chromatography. This enzyme was recognised by antibodies to the native nNOS and showed a similar degree of inhibition by arginine analogs as the native nNOS. The majority of the NOS synthesised had accumulated as insoluble "inclusion-body" material. The purification of recombinant nNOS from insect cells should facilitate characterisation of neuronal NO synthase.
Charles
I G
IG
Wellcome Research Laboratories, Langley Court, Beckenham, Kent, UK.
Chubb
A
A
Gill
R
R
Clare
J
J
Lowe
P N
PN
Holmes
L S
LS
Page
M
M
Keeling
J G
JG
Moncada
S
S
Riveros-Moreno
V
V
D000594:Amino Acid Oxidoreductases / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D000818:Animals; D016367:Baculoviridae; D001483:Base Sequence; D015153:Blotting, Western; D001921:Brain / Q000201:enzymology*; D002460:Cell Line; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D015723:Gene Library; D008969:Molecular Sequence Data; D009036:Moths; D009474:Neurons / Q000201:enzymology*; D019001:Nitric Oxide Synthase; D016376:Oligonucleotides, Antisense; D016133:Polymerase Chain Reaction; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D015183:Restriction Mapping; D014162:Transfection
D017931:DNA Primers; D018076:DNA, Complementary; D016376:Oligonucleotides, Antisense; D011994:Recombinant Proteins; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases