ArticleInternational journal of molecular sciences2019
Development and Optimization of Expression, Purification, and ATPase Assay of KaiC for Medium-Throughput Screening of Circadian Clock Mutants in Cyanobacteria.
Article in International journal of molecular sciences, 2019. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 7 papers.
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The abstract states no effect estimate the extractor could read, or names no intervention and outcome on the map, so this paper lights no cell and moves no belief. It is still indexed, cited and linked below.
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Trials whose registry record cites this paper, or whose number appears in the abstract. A trial that started after this paper was published is citing it as background, not reporting it.
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Who cites it
7 citing papers in PubMed, 13 citations in OpenAlex.
- Structure-function relationship of KaiC around dawn.Biophysics and physicobiology · 2024Article
- Insights about circadian clock in glioma: From molecular pathways to therapeutic drugs.CNS neuroscience & therapeutics · 2022Review
- Regulation mechanisms of the dual ATPase in KaiC.Proceedings of the National Academy of Sciences of the United States of America · 2022Article
- Elucidation of master allostery essential for circadian clock oscillation in cyanobacteria.Science advances · 2022Article
- Slow and temperature-compensated autonomous disassembly of KaiB-KaiC complex.Biophysics and physicobiology · 2022Article
- Orchestration of Circadian Timing by Macromolecular Protein Assemblies.Journal of molecular biology · 2020Review
- Biophysical research in Okazaki, Japan.Biophysical reviews · 2020Review
Corrections and comments
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Authors and funding
6 authors at 2 institutions in 1 country.
Funding
Abstract
The slow but temperature-insensitive adenosine triphosphate (ATP) hydrolysis reaction in KaiC is considered as one of the factors determining the temperature-compensated period length of the cyanobacterial circadian clock system. Structural units responsible for this low but temperature-compensated ATPase have remained unclear. Although whole-KaiC scanning mutagenesis can be a promising experimental strategy, producing KaiC mutants and assaying those ATPase activities consume considerable time and effort. To overcome these bottlenecks for in vitro screening, we optimized protocols for expressing and purifying the KaiC mutants and then designed a high-performance liquid chromatography system equipped with a multi-channel high-precision temperature controller to assay the ATPase activity of multiple KaiC mutants simultaneously at different temperatures. Through the present protocol, the time required for one KaiC mutant is reduced by approximately 80% (six-fold throughput) relative to the conventional protocol with reasonable reproducibility. For validation purposes, we picked up three representatives from 86 alanine-scanning KaiC mutants preliminarily investigated thus far and characterized those clock functions in detail.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.