Detecting KaiC phosphorylation rhythms of the cyanobacterial circadian oscillator in vitro and in vivo

Authors

Yongick Kim, Marshall UniversityFollow
Joseph S. Boyd
Javier Espinosa
Susan S. Golden

Document Type

Article

Publication Date

1-2015

Abstract

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 phosphorylation states of KaiC and other phosphorylated proteins from in vivo samples.

Comments

This is the Authors’ manuscript published in final edited form in Methods in Enzymology. The version of record is available from the publisher at https://doi.org/10.1016%2Fbs.mie.2014.10.003. Copyright © 2015 Elsevier Inc. All rights reserved.

Recommended Citation

Kim YI, Boyd JS, Espinosa J, Golden SS. Detecting KaiC phosphorylation rhythms of the cyanobacterial circadian oscillator in vitro and in vivo. Methods Enzymol. 2015;551:153-73. doi: 10.1016/bs.mie.2014.10.003.