Development and Optimization of Expression, Purification, and ATPase Assay of KaiC for Medium-Throughput Screening of Circadian Clock Mutants in Cyanobacteria
Abstract
:1. Introduction
2. Results
2.1. Optimization of the Expression and Purification of KaiC
2.2. Development of the HPLC System Equipped with a Multi-Channel High-Precision Temperature Controller
2.3. Throughput, Performance, and Reproducibility of the Protocol
2.4. Detailed Characterizations of Representative ATPase Mutants of KaiC
3. Discussion
4. Materials and Methods
4.1. Expressions and Purifications
4.2. Development of Temperature-Controlled Sample Tables
4.3. ATPase Measurements
4.4. KaiC Phosphorylation/Dephosphorylation Cycle
4.5. In Vivo Bioluminsescence Assay
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
HPLC | high-performance liquid chromatography |
ATP | adenosine triphosphate |
ADP | adenosine diphosphate |
PTO | post-translational oscillation |
TTO | transcriptional and translational oscillation |
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KaiC | In Vitro ATPase Activity 1,2 | In Vitro Phosphorylation Cycle 1,3 | In Vivo Bioluminescence Rhythm 1 | |||
---|---|---|---|---|---|---|
(d−1) 4 | Q10 | Period (h) 4 | Q10 | Period (h) 5 | Q106 | |
WT | 13.4 ± 0.9 | 1.01 ± 0.04 | 23.1 ± 0.3 | 1.09 ± 0.00 | 24.8 ± 0.2 | 1.05 ± 0.01 (0.99 ± 0.01) |
P37A | 12.9 ± 1.6 * | 1.03 ± 0.02 * | 24.6 ± 0.3 *** | 1.08 ± 0.00 *** | 28.1 ± 0.1 **** | 1.01 ± 0.01 *** (0.93 ± 0.01 ***) |
T228A | 9.8 ± 0.9 *** | 1.18 ± 0.06 ** | 30.2 ± 0.4 **** | 1.15 ± 0.01 *** | 30.2 ± 0.2 **** | 1.17 ± 0.03 ** (0.94 ± 0.03 *) |
C306A | 15.9 ± 2.2 * | 1.14 ± 0.05 ** | 19.6 ± 0.1 **** | 1.14 ± 0.02 ** | 20.8 ± 0.0 **** | 1.09 ± 0.01 *** (1.05 ± 0.01 ****) |
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Ouyang, D.; Furuike, Y.; Mukaiyama, A.; Ito-Miwa, K.; Kondo, T.; Akiyama, S. Development and Optimization of Expression, Purification, and ATPase Assay of KaiC for Medium-Throughput Screening of Circadian Clock Mutants in Cyanobacteria. Int. J. Mol. Sci. 2019, 20, 2789. https://doi.org/10.3390/ijms20112789
Ouyang D, Furuike Y, Mukaiyama A, Ito-Miwa K, Kondo T, Akiyama S. Development and Optimization of Expression, Purification, and ATPase Assay of KaiC for Medium-Throughput Screening of Circadian Clock Mutants in Cyanobacteria. International Journal of Molecular Sciences. 2019; 20(11):2789. https://doi.org/10.3390/ijms20112789
Chicago/Turabian StyleOuyang, Dongyan, Yoshihiko Furuike, Atsushi Mukaiyama, Kumiko Ito-Miwa, Takao Kondo, and Shuji Akiyama. 2019. "Development and Optimization of Expression, Purification, and ATPase Assay of KaiC for Medium-Throughput Screening of Circadian Clock Mutants in Cyanobacteria" International Journal of Molecular Sciences 20, no. 11: 2789. https://doi.org/10.3390/ijms20112789
APA StyleOuyang, D., Furuike, Y., Mukaiyama, A., Ito-Miwa, K., Kondo, T., & Akiyama, S. (2019). Development and Optimization of Expression, Purification, and ATPase Assay of KaiC for Medium-Throughput Screening of Circadian Clock Mutants in Cyanobacteria. International Journal of Molecular Sciences, 20(11), 2789. https://doi.org/10.3390/ijms20112789