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Expression, purification and characterization of the secreted luciferase of the copepod Metridia longa from Sf9 insect cells [Text] / G. A. Stepanyuk [et al.] // Protein Expr. Purif. - 2008. - Vol. 61, Is. 2. - P142-148, DOI 10.1016/j.pep.2008.05.013. - Cited References: 34. - This work was supported by the National Institutes of Health (Grant 1P50 GM62407), University of Georgia Research Foundation and Georgia Research Alliance, the Russian Foundation for Basic Research and Taiwan National Science Council (Grant 06-0489502) and the program for "Molecular and Cellular Biology" of Russian Academy of Sciences. . - ISSN 1046-5928

РУБ Biochemical Research Methods + Biochemistry & Molecular Biology + Biotechnology & Applied Microbiology
Рубрики:
VARGULA-HILGENDORFII LUCIFERASE
   CRYSTAL-STRUCTURE
   RENILLA-RENIFORMIS
   GAUSSIA LUCIFERASE
   BIOLUMINESCENT REPORTER
   OBELIN BIOLUMINESCENCE
   ANGSTROM RESOLUTION
   MAMMALIAN-CELLS
   GENE-EXPRESSION
   IN-VIVO
Аннотация: Metridia luciferase is a secreted luciferase from a marine copepod and uses coelenterazine as a substrate to produce a blue bioluminescence This luciferase has been successfully applied as a bioluminescent reporter in mammalian cells. The main advantage of secreted luciferase as a reporter is the capability of measuring intracellular events without destroying the cells or tissues and this property is well suited for development of high throughput screening technologies. However because Metridia luciferase is a Cys-rich protein, Escherichia coli expression systems produce an incorrectly folded protein, hindering its biochemical characterization and application for development of in vitro bioluminescent assays. Here we report the successful expression of Metridia luciferase with its signal peptide for secretion, in insect (Sf9) cells using the baculovirus expression system. Functionally active luciferase secreted by insect cells into the culture media has been efficiently purified with a yield of high purity protein of 2-3mg/L This Metridia luciferase expressed in the insect cell system is a monomeric protein showing 3.5-fold greater bioluminescence activity than luciferase expressed and purified from E. coli. The near coincidence of the experimental mass of Metridia luciferase purified from insect cells with that calculated from amino acid sequence. indicates that luciferase does not undergo post-translational modifications such as phosphorylation or glycosylation and also, the cleavage site of the signal peptide for secretion is at VQA-KS, as predicted from sequence analysis. (c) 2008 Elsevier Inc. All rights reserved.

Держатели документа:
[Stepanyuk, Galina A.
Markova, Svetlana V.
Vysotski, Eugene S.] Russian Acad Sci, Photobiol Lab, Inst Biophys, Siberian Branch, Krasnoyarsk 660036, Russia
[Stepanyuk, Galina A.
Xu, Hao
Wu, Chia-Kuei
Lee, John
Vysotski, Eugene S.
Wang, Bi-Cheng] Univ Georgia, Dept Biochem & Mol Biol, Athens, GA 30602 USA
ИБФ СО РАН : 660036, Красноярск, Академгородок, д. 50, стр. 50

Доп.точки доступа:
Stepanyuk, G.A.; Xu, H...; Wu, C.K.; Markova, S.V.; Lee, J...; Vysotski, E.S.; Wang, B.C.

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Cloning and expression of cDNA for a luciferase from the marine copepod Metridia longa - A novel secreted bioluminescent reporter enzyme [Text] / S. V. Markova [et al.] // J. Biol. Chem. - 2004. - Vol. 279, Is. 5. - P3212-3217, DOI 10.1074/jbc.M309639200. - Cited References: 37 . - ISSN 0021-9258

РУБ Biochemistry & Molecular Biology
Рубрики:
VARGULA-HILGENDORFII LUCIFERASE
   GREEN FLUORESCENT PROTEIN
   GENE-EXPRESSION
   FIREFLY LUCIFERASE
   PROMOTER ACTIVITY
   MAMMALIAN-CELLS
   RECEPTOR
   CANCER
   PHOTOPROTEINS
   LUMINESCENCE
Аннотация: Metridia longa is a marine copepod from which a blue bioluminescence originates as a secretion from epidermal glands in response to various stimuli. We demonstrate that Metridia luciferase is specific for coelenterazine to produce blue light (lambda(max)=480 nm). Using an expression cDNA library and functional screening, we cloned and sequenced the cDNA encoding the Metridia luciferase. The cDNA is an 897-bp fragment with a 656-bp open reading frame, which encodes a 219-amino acid polypeptide with a molecular weight of 23,885. The polypeptide contains an N-terminal signal peptide of 17 amino acid residues for secretion. On expression of the Metridia luciferase gene in mammalian Chinese hamster ovary cells the luciferase is detected in the culture medium confirming the existence of a naturally occurring signal peptide for secretion in the cloned luciferase. The novel secreted luciferase was tested in a practical assay application in which the activity of A2a and NPY2 G-protein-coupled receptors was detected. These results clearly suggest that the secreted Metridia luciferase is well suited as a reporter for monitoring gene expression and, in particular, for the development of novel ultra-high throughput screening technologies.

Держатели документа:
Russian Acad Sci, Siberian Branch, Inst Biophys, Photobiol Lab, Krasnoyarsk 660036, Russia
Bayer AG, Pharma Res Mol Screening Technol, D-42096 Wuppertal, Germany
ИБФ СО РАН : 660036, Красноярск, Академгородок, д. 50, стр. 50

Доп.точки доступа:
Markova, S.V.; Golz, S...; Frank, L.A.; Kalthof, B...; Vysotski, E.S.

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Large-scale, high-throughput validation of short hairpin RNA sequences for RNA interference [Text] / L. H. Lamarcq [et al.] // J. Biomol. Screen. - 2006. - Vol. 11, Is. 3. - P236-246, DOI 10.1177/1087057105284342. - Cited References: 50 . - ISSN 1087-0571

РУБ Biochemical Research Methods + Biotechnology & Applied Microbiology + Chemistry, Analytical
Рубрики:
ENZYME FRAGMENT COMPLEMENTATION
   ORFEOME VERSION 1.1
   SMALL NUCLEAR-RNA
   MAMMALIAN-CELLS
   GENE-EXPRESSION
   SIRNA SEQUENCES
   POLYMERASE-III
   SELECTION
   TRANSCRIPTION
   MICROARRAYS
Кл.слова (ненормированные):
RNAi -- high-throughput screening -- target validation -- shRNA -- reporter assay
Аннотация: (shRNAs) is described. Using this approach, 464 shRNAs auainst 116 different genes were screened for knockdown efficacy, enabling rapid identification of effective shRNAS against 74 genes. Statistical analysis of the effects of various criteria on the activity of the shRNAs confirmed that some of the rules thought to govern small interfering RNA (siRNA) activity also apply to shRNAs. These include moderate GC content, absence of internal hairpins, and asymmetric thermal stability. However, the authors did not find strong support for position-specific rules. In addition, analysis of the data suggests that not all genes are equally susceptible to RNA interference (RNAi).

Держатели документа:
Clontech Labs, Mountain View, CA 94043 USA
BD Biosci Pharmingen, La Jolla, CA USA
Bayer Healthcare AG, Pharma Res, Mol Screening Technol, Wuppertal, Germany
Russian Acad Sci, Inst Biophys, Photobiol Lab, Siberian Branch, Krasnoyarsk, Russia
ИБФ СО РАН : 660036, Красноярск, Академгородок, д. 50, стр. 50

Доп.точки доступа:
Lamarcq, L.H.; Scherer, B.J.; Phelan, M.L.; Kalnine, N.N.; Nguyen, Y.H.; Kabakova, T...; Chen, X.Y.; Tan, M...; Chang, C...; Berlon, C...; Campos-Gonzalez, R...; Gao, G.J.; Golz, S...; Vysotski, E.S.; Farmer, A.A.

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