Тип публикации: статья из журнала
Год издания: 2012
Идентификатор DOI: 10.1111/j.1742-4658.2012.08476.x
Ключевые слова: bioluminescence, calcium, coelenterazine, luciferase, mammalian expression, bioluminescence, calcium, coelenterazine, luciferase, mammalian expression, berovin, complementary DNA, photoprotein, unclassified drug, article, bacterial cell, binding site, bioluminescence, controlled study, DNA sequence, enzyme activity, Escherichia coli, molecular cloning, nucleotide sequence, priority journal, protein expression, protein isolation, protein purification, sequence analysis, Amino Acid Sequence, Animals, Binding Sites, Calcium, CHO Cells, Cloning, Molecular, Cricetinae, Ctenophora, Hydrogen-Ion Concentration, Kinetics, Light, Luciferases, Luminescent Measurements, Luminescent Proteins, Molecular Sequence Data, Recombinant Proteins, Sequence Homology, Amino Acid, Beroe abyssicola, Ctenophora (coelenterates), Escherichia coli, Mammalia, Scyphozoa
Аннотация: Light-sensitive Ca2+-regulated photoproteins are responsible for the bright bioluminescence of ctenophores. Using functional screening, four full-size cDNA genes encoding the same 208-amino-acid polypeptide were isolated from two independent cDNA libraries prepared from two Beroe abyssicola specimens. Sequence analysis revealed three canonical EF-hand calcium-binding sites characteristic of Ca2+-regulated photoproteins, but a very low degree of sequence identity (2729%) with aequorin-type photoproteins, despite functional similarities. Recombinant berovin was expressed in Escherichia coli cells, purified, converted to active photoprotein and characterized. Active berovin has absorption maxima at 280 and 437 nm. The Ca2+-discharged protein loses visible absorption, but exhibits a new absorption maximum at 335 nm. The berovin bioluminescence is blue (?max = 491 nm) and a change in pH over the range 6.09.5 has no significant effect on the light emission spectrum. By contrast, the fluorescence of Ca2+-discharged protein (?ex = 350 nm) is pH sensitive: at neutral pH the maximum is at 420 nm and at alkaline pH there are two maxima at 410 and 485 nm. Like native ctenophore photoproteins, recombinant berovin is also inactivated by light. The Ca2+ concentrationeffect curve is a sigmoid with a slope on a loglog plot of similar to 2.5. Although this curve for berovin is very similar to those obtained for obelin and aequorin, there are evident distinctions: berovin responds to calcium changes at lower concentrations than jellyfish photoproteins and its Ca2+-independent luminescence is low. Recombinant berovin was successfully expressed in mammalian cells, thereby demonstrating potential for monitoring intracellular calcium.
Журнал: FEBS JOURNAL
Выпуск журнала: Vol. 279, Is. 5
Номера страниц: 856-870
ISSN журнала: 1742464X
Место издания: MALDEN
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