Abstract

The cyanobacterial circadian clock is the most well-understood and simplest biological time-keeping system. Its oscillator consists of three proteins: KaiA, KaiB, and KaiC. When combined together in a test tube, the Kai proteins produce a free-running 24-h cycle of rhythmic auto-phosphorylation and auto-dephosphorylation. To generate a robust circadian rhythm of the in vitro reaction mixture, KaiC, the core oscillator protein, must be purified with an untraditional approach, since even the smallest amount of impurity can hinder its post-translational activities. Until recently, series of fast protein liquid chromatography (FPLC) columns (glutathione S-transferase (GST), anion exchange (Q), and desalting columns) have been used to purify the oscillator proteins, often requiring laborious elution processes. Although the common methodology has already been established, whether the purified KaiC can produce robust oscillations remains to be verified. Here we emphasize the significance of eliminating the Q step and lengthening the step of removing the non-specifically bound impurities on the GST column for generating a rhythmic KaiC phosphorylation in vitro. These findings demonstrate the potential for shortening the amount of time and effort it takes to purify proteins without compromising its quality.

中文翻译：

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GST融合的蓝细菌中央振荡器蛋白KaiC的纯化

摘要

蓝细菌生物钟是最容易理解和最简单的生物计时系统。它的振荡器由三种蛋白质组成：KaiA，KaiB和KaiC。当在试管中结合在一起时，Kai蛋白会产生有规律的24小时自律性自磷酸化和自脱磷酸化循环。为了产生体外反应混合物的稳健的昼夜节律，必须使用非传统方法纯化核心振荡器蛋白KaiC，因为即使最少量的杂质也会阻碍其翻译后活性。直到最近，一系列快速蛋白质液相色谱（FPLC）柱（谷胱甘肽S-转移酶（GST），阴离子交换（Q）和脱盐柱）已用于纯化振荡器蛋白，通常需要费力的洗脱过程。尽管已经建立了通用方法，但是纯化的KaiC是否可以产生稳定的振荡仍有待验证。在这里，我们强调消除Q步骤并延长去除GST色谱柱上非特异性结合杂质的步骤的重要性，以在体外产生有节奏的KaiC磷酸化。这些发现表明，在不影响蛋白质质量的前提下，可以缩短纯化蛋白质所需的时间和精力。