Photobiology Lab, Institute of Biophysics, Russian Academy of Sciences, Krasnoyarsk, Russia.
FEBS J. 2012 Mar;279(5):856-70. doi: 10.1111/j.1742-4658.2012.08476.x. Epub 2012 Feb 10.
Light-sensitive Ca(2+) -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 Ca(2+) -regulated photoproteins, but a very low degree of sequence identity (27-29%) 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 Ca(2+) -discharged protein loses visible absorption, but exhibits a new absorption maximum at 335 nm. The berovin bioluminescence is blue (λ(max) = 491 nm) and a change in pH over the range 6.0-9.5 has no significant effect on the light emission spectrum. By contrast, the fluorescence of Ca(2+) -discharged protein (λ(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 Ca(2+) concentration-effect curve is a sigmoid with a slope on a log-log plot of ∼ 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 Ca(2+) -independent luminescence is low. Recombinant berovin was successfully expressed in mammalian cells, thereby demonstrating potential for monitoring intracellular calcium. Database The nucleotide sequences have been deposited in the GenBankTM/EBI Data Bank with accession numbers: apoberovin cDNA genes, JN673813 (BA1), JN673814 (BA2), JN673815 (BA3), JN673816 (BA4); fragment 18S rRNA, JN673817 (BA-rRNA5).
发光敏感的 Ca(2+)调节光蛋白是栉水母生物发光的主要原因。通过功能筛选，从两个独立的 cDNA 文库中分离出四个全长 cDNA 基因，分别编码相同的 208 个氨基酸多肽。序列分析表明，这四个基因都含有三个典型的 EF 手钙离子结合位点，这是 Ca(2+)调节光蛋白的特征，但与水母发光蛋白的序列同一性很低（27-29%），尽管功能相似。重组 berovin 在大肠杆菌细胞中表达、纯化、转化为活性光蛋白并进行了表征。活性 berovin 在 280nm 和 437nm 处有吸收最大值。钙离子释放的蛋白失去可见光吸收，但在 335nm 处显示新的吸收最大值。berovin 生物发光呈蓝色（λ(max) = 491nm），pH 值在 6.0-9.5 范围内变化对发光光谱没有显著影响。相比之下，钙离子释放蛋白的荧光（λ(ex) = 350nm）对 pH 值敏感：在中性 pH 值下，最大值为 420nm，在碱性 pH 值下有两个最大值分别在 410nm 和 485nm。与天然栉水母光蛋白一样，重组 berovin 也会被光失活。Ca(2+)浓度效应曲线是一个 S 形曲线，在对数-对数图上的斜率约为 2.5。尽管 berovin 的曲线与水母发光蛋白的曲线非常相似，但存在明显的区别：berovin 对钙离子变化的反应浓度低于水母发光蛋白，其 Ca(2+) 非依赖性发光较低。重组 berovin 成功地在哺乳动物细胞中表达，从而证明了监测细胞内钙离子的潜力。
该核苷酸序列已在 GenBankTM/EBI 数据银行中注册，登录号分别为：apoberovin cDNA 基因，JN673813（BA1）、JN673814（BA2）、JN673815（BA3）、JN673816（BA4）；18S rRNA 片段，JN673817（BA-rRNA5）。
