Cloning and expression of a rat neuronal nitric oxide synthase coding sequence in a baculovirus/insect cell system.
Charles, IG
Chubb, A
Gill, R
Clare, J
Lowe, PN
Holmes, LS
Page, M
Keeling, JG
Moncada, S
Riveros-Moreno, V
- Publication Type:
- Journal Article
- Citation:
- Biochem Biophys Res Commun, 1993, 196 (3), pp. 1481 - 1489
- Issue Date:
- 1993-11-15
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2009002909OK.pdf | 467.62 kB | Adobe PDF |
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Full metadata record
Field | Value | Language |
---|---|---|
dc.contributor.author | Charles, IG | en_US |
dc.contributor.author | Chubb, A | en_US |
dc.contributor.author | Gill, R | en_US |
dc.contributor.author | Clare, J | en_US |
dc.contributor.author | Lowe, PN | en_US |
dc.contributor.author | Holmes, LS | en_US |
dc.contributor.author | Page, M | en_US |
dc.contributor.author | Keeling, JG | en_US |
dc.contributor.author | Moncada, S | en_US |
dc.contributor.author | Riveros-Moreno, V | en_US |
dc.date.issued | 1993-11-15 | en_US |
dc.identifier.citation | Biochem Biophys Res Commun, 1993, 196 (3), pp. 1481 - 1489 | en_US |
dc.identifier.issn | 0006-291X | en_US |
dc.identifier.uri | http://hdl.handle.net/10453/13366 | |
dc.description.abstract | 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. | en_US |
dc.language | eng | en_US |
dc.relation.ispartof | Biochem Biophys Res Commun | en_US |
dc.relation.isbasedon | 10.1006/bbrc.1993.2419 | en_US |
dc.subject.classification | Biochemistry & Molecular Biology | en_US |
dc.subject.mesh | Brain | en_US |
dc.subject.mesh | Neurons | en_US |
dc.subject.mesh | Cell Line | en_US |
dc.subject.mesh | Animals | en_US |
dc.subject.mesh | Rats | en_US |
dc.subject.mesh | Moths | en_US |
dc.subject.mesh | Baculoviridae | en_US |
dc.subject.mesh | Amino Acid Oxidoreductases | en_US |
dc.subject.mesh | Recombinant Proteins | en_US |
dc.subject.mesh | Oligonucleotides, Antisense | en_US |
dc.subject.mesh | DNA, Complementary | en_US |
dc.subject.mesh | DNA Primers | en_US |
dc.subject.mesh | Blotting, Western | en_US |
dc.subject.mesh | Electrophoresis, Polyacrylamide Gel | en_US |
dc.subject.mesh | Restriction Mapping | en_US |
dc.subject.mesh | Cloning, Molecular | en_US |
dc.subject.mesh | Transfection | en_US |
dc.subject.mesh | Polymerase Chain Reaction | en_US |
dc.subject.mesh | Gene Expression | en_US |
dc.subject.mesh | Base Sequence | en_US |
dc.subject.mesh | Gene Library | en_US |
dc.subject.mesh | Molecular Sequence Data | en_US |
dc.subject.mesh | Nitric Oxide Synthase | en_US |
dc.title | Cloning and expression of a rat neuronal nitric oxide synthase coding sequence in a baculovirus/insect cell system. | en_US |
dc.type | Journal Article | |
utslib.citation.volume | 3 | en_US |
utslib.citation.volume | 196 | en_US |
utslib.location.activity | United States | en_US |
utslib.for | 0601 Biochemistry and Cell Biology | en_US |
utslib.for | 0304 Medicinal and Biomolecular Chemistry | en_US |
utslib.for | 1101 Medical Biochemistry and Metabolomics | en_US |
dc.location.activity | ISI:A1993MG31000067 | en_US |
pubs.embargo.period | Not known | en_US |
pubs.organisational-group | /University of Technology Sydney | |
pubs.organisational-group | /University of Technology Sydney/Faculty of Science | |
pubs.organisational-group | /University of Technology Sydney/Strength - ithree - Institute of Infection, Immunity and Innovation | |
utslib.copyright.status | closed_access | |
pubs.issue | 3 | en_US |
pubs.publication-status | Published | en_US |
pubs.volume | 196 | en_US |
Abstract:
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.
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