Development and Optimization of Expression, Purification, and ATPase Assay of KaiC for Medium-Throughput Screening of Circadian Clock Mutants in Cyanobacteria.

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

SUBMITTER: Ouyang D 

PROVIDER: S-EPMC6600144 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature