Detecting KaiC Phosphorylation Rhythms of the Cyanobacterial Circadian Oscillator In Vitro and In Vivo
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Título: | Detecting KaiC Phosphorylation Rhythms of the Cyanobacterial Circadian Oscillator In Vitro and In Vivo |
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Autor/es: | Kim, Yong-Ick | Boyd, Joseph S. | Espinosa, Javier | Golden, Susan S. |
Grupo/s de investigación o GITE: | Transducción de Señales en Bacterias |
Centro, Departamento o Servicio: | Universidad de Alicante. Departamento de Fisiología, Genética y Microbiología |
Palabras clave: | Biological clock | Circadian rhythms | Cyanobacteria | KaiC | FPLC | Oscillatory reaction | Phos-tag | Protein purification | Phosphorylation | Synechococcus elongatus |
Área/s de conocimiento: | Genética |
Fecha de publicación: | 2015 |
Editor: | Elsevier |
Cita bibliográfica: | Methods in Enzymology. 2015, 551: 153-173. doi:10.1016/bs.mie.2014.10.003 |
Resumen: | 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. |
Patrocinador/es: | This work was supported by National Institutes of Health Grant R01GM062419 (to S. S. G.). |
URI: | http://hdl.handle.net/10045/58327 |
ISSN: | 0076-6879 (Print) | 1557-7988 (Online) |
DOI: | 10.1016/bs.mie.2014.10.003 |
Idioma: | eng |
Tipo: | info:eu-repo/semantics/article |
Derechos: | © 2015 Elsevier Inc. |
Revisión científica: | si |
Versión del editor: | http://dx.doi.org/10.1016/bs.mie.2014.10.003 |
Aparece en las colecciones: | INV - TSB - Artículos de Revistas |
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2015_Kim_etal_MethEnzymology_final.pdf | Versión final (acceso restringido) | 279,77 kB | Adobe PDF | Abrir Solicitar una copia |
2015_Kim_etal_MethEnzymology_preprint.pdf | Preprint (acceso abierto) | 762,14 kB | Adobe PDF | Abrir Vista previa |
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