Тип публикации: статья из журнала
Год издания: 2015
Идентификатор DOI: 10.1016/j.bbrc.2014.12.082
Ключевые слова: Bioluminescence, Coelenterazine, Copepod luciferase, Mammalian expression, Real-time imaging, complementary DNA, cysteine, luciferase, animal cell, Article, baculovirus expression system, chemical bond, cloning, copepod, culture medium, gene, gene frequency, genetic code, human, human cell, insect cell, mammal cell, Metridia longa, nonhuman, nucleotide sequence, priority journal, protein analysis, protein folding, protein purification, protein secretion, sensitivity analysis, amino acid sequence, animal, cell inclusion, chemistry, enzymology, genetics, HEK293 cell line, kinetics, luminescence, metabolism, molecular cloning, molecular genetics, molecular weight, sequence alignment, SF9 cell line, time, Bacteria (microorganisms), Copepoda, Hexapoda, Mammalia, Metridia luciferase, Animals, Cloning, Molecular, HEK293 Cells, Humans, Inclusion Bodies, Luciferases, Luminescent Measurements, Molecular Sequence Data, Sf9 Cells, Time Factors
Аннотация: Coelenterazine-dependent copepod luciferases containing natural signal peptide for secretion are a very convenient analytical tool as they enable monitoring of intracellular events with high sensitivity, without destroying cells or tissues. This property is well suited for application in biomedical research and development of cell-based assays for high throughput screening. We report the cloning of cDNA gene encoding a novel secreted non-allelic 16.5-kDa isoform (MLuc7) of Metridia longa luciferase, which, in fact, is the smallest natural luciferase of known for today. Despite the small size, isoform contains 10 conservative Cys residues suggesting the presence of up to 5 S-S bonds. This hampers the efficient production of functionally active recombinant luciferase in bacterial expression systems. With the use of the baculovirus expression system, we produced substantial amounts of the proper folded MLuc7 luciferase with a yield of similar to 3 mg/L of a high purity protein. We demonstrate that MLuc7 produced in insect cells is highly active and extremely thermostable, and is well suited as a secreted reporter when expressed in mammalian cells ensuring higher sensitivity of detection as compared to another Metridia luciferase isoform (MLuc164) which is widely employed in real-time imaging. (C) 2014 Elsevier Inc. All rights reserved.
Журнал: BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Выпуск журнала: Vol. 457, Is. 1
Номера страниц: 77-82
ISSN журнала: 0006291X
Место издания: SAN DIEGO
Издатель: ACADEMIC PRESS INC ELSEVIER SCIENCE
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