Purification of GST-Fused Cyanobacterial Central Oscillator Protein KaiC

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.