Watkins N J, Campbell A K
Department of Medical Biochemistry, University of Wales College of Medicine, Heath Park, Cardiff, U.K.
Biochem J. 1993 Jul 1;293 ( Pt 1)(Pt 1):181-5. doi: 10.1042/bj2930181.
cDNA coding for the Ca(2+)-activated photoprotein aequorin from the jellyfish Aequorea victoria has been engineered to investigate the role of the C-terminal proline residue in bioluminescence. Recombinant aequorin proteins were synthesized by PCR followed by in vitro transcription/translation, and characterized by specific activity, stability, and affinity for coelenterazine. The C-terminal proline residue of aequorin was shown to be essential for the long-term stability of the bound coelenterazine. Aequorin minus proline had only 1% of the specific activity of the wild-type after 2 h, and was virtually inactive after 18 h. The instability of this variant was further demonstrated by re-activating with a coelenterazine analogue (epsilon-coelenterazine), where maximum reactivation was reached in 15 min, and the luminescent activity was almost completely abolished within 3 h. Replacement of the C-terminal proline residue with histidine or glutamic acid decreased the specific activity to 10 and 19% of that of the wild-type respectively. However these variants were also unstable, having t1/2 values of 2.4 h and 2.3 h respectively. Enhancement of the Ca(2+)-independent light emission when proline was replaced by histidine confirmed the stabilizing role of the C-terminal proline. No significant effect of removal of the C-terminal proline was detected on the affinity for coelenterazine.
为了研究水母维多利亚多管水母中编码钙离子激活光蛋白水母发光蛋白的cDNA中C端脯氨酸残基在生物发光中的作用，对其进行了工程改造。通过聚合酶链反应（PCR）合成重组水母发光蛋白，随后进行体外转录/翻译，并通过比活性、稳定性和对腔肠素的亲和力对其进行表征。结果表明，水母发光蛋白的C端脯氨酸残基对于结合的腔肠素的长期稳定性至关重要。去除脯氨酸的水母发光蛋白在2小时后比活性仅为野生型的1%，18小时后几乎无活性。用腔肠素类似物（ε-腔肠素）重新激活进一步证明了该变体的不稳定性，在15分钟内达到最大重新激活，发光活性在3小时内几乎完全消失。用组氨酸或谷氨酸取代C端脯氨酸残基分别使比活性降至野生型的10%和19%。然而，这些变体也不稳定，半衰期分别为2.4小时和2.3小时。当脯氨酸被组氨酸取代时，钙离子非依赖性发光增强，证实了C端脯氨酸的稳定作用。未检测到去除C端脯氨酸对腔肠素亲和力的显著影响。
