Detecting KaiC phosphorylation rhythms of the cyanobacterial circadian oscillator in vitro and in vivo.
Detecting KaiC phosphorylation rhythms of the cyanobacterial circadian oscillator in vitro and in vivo.
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DOI：
10.1016/bs.mie.2014.10.003
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发表时间：
2015
影响因子：
--
通讯作者：
Golden SS
中科院分区：
文献类型：
--
作者：
Kim YI;Boyd JS;Espinosa J;Golden SS
蓝藻生物钟的中心振荡器是独特的，其组成部分的生化简单性和振荡的鲁棒性。振荡器由三种蓝藻蛋白质KaiA、KaiB和KaiC组成。如果将这三种蛋白质的非常纯的制剂以正确的比例与ATP和MgCl 2混合在试管中，KaiC的磷酸化状态将随着昼夜节律周期振荡，并且这些状态可以简单地通过SDS-PAGE进行分析。蛋白质的纯度对于获得稳定的振荡至关重要。污染的蛋白酶将通过Kai蛋白质的降解破坏振荡，并且ATP酶将通过消耗ATP减弱鲁棒性。在这里，我们提供了一个详细的协议，以获得纯的重组蛋白从大肠杆菌构建一个强大的蓝藻昼夜节律振荡器在体外。此外，我们提出了一个协议，有利于分析的磷酸化状态KaiC和其他磷酸化的蛋白质在体内样品。
The central oscillator of the cyanobacterial circadian clock is unique in the biochemical simplicity of its components and the robustness of the oscillation. The oscillator is composed of three cyanobacterial proteins, KaiA, KaiB, and KaiC. If very pure preparations of these three proteins are mixed in a test tube in the right proportions and with ATP and MgCl2, the phosphorylation states of KaiC will oscillate with a circadian period and these states can be analyzed simply by SDS-PAGE. The purity of the proteins is critical for obtaining robust oscillation. Contaminating proteases will destroy oscillation by degradation of Kai proteins, and ATPases will attenuate robustness by consumption of ATP. Here, we provide a detailed protocol to obtain pure recombinant proteins from Escherichia coli to construct a robust cyanobacterial circadian oscillator in vitro. In addition, we present a protocol that facilitates analysis of phosphoryation states of KaiC and other phosphorylated proteins from in vivo samples.