Abstract
Spiroplasma is a genus of wall-less helical bacteria with swimming motility unrelated to conventional types of bacterial motility machinery, such as flagella and pili. The swimming of Spiroplasma is suggested to be driven by five classes of MreB (MreB1-MreB5), which are members of the actin superfamily. In vitro studies of Spiroplasma MreBs have recently been conducted to evaluate their activities, such as ATPase, which is essential for the polymerization dynamics among classic actin superfamily proteins. In this chapter, we describe methods of purification and Pi release measurement of Spiroplasma MreBs using column chromatography and absorption spectroscopy with the molecular probe, 2-amino-6-mercapto-7-methylpurine riboside (MESG). Of note, the methods described here are applicable to other proteins that possess NTPase activity.
Key words
- Bacterial actin cytoskeleton
- E. coli expression system
- Recombinant protein
- Ni2+-NTA affinity chromatography
- Gel filtration
- Pi release assay
- Absorption spectroscopy
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Acknowledgments
This study was supported by Grants-in-Aid for Scientific Research (A and C) (MEXT KAKENHI, Grant Numbers JP17H01544 to MM and JP20K06591 to IF), JST CREST (Grant Number JPMJCR19S5 to MM), the Research Foundation of Opto-Science and Technology to IF, and the Osaka City University (OCU) Strategic Research Grant 2019 to IF. DT is a recipient of the Research Fellowship of the Japan Society for the Promotion of Science (22J10345).
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Takahashi, D., Fujiwara, I., Miyata, M. (2023). Purification and ATPase Activity Measurement of Spiroplasma MreB. In: Minamino, T., Miyata, M., Namba, K. (eds) Bacterial and Archaeal Motility. Methods in Molecular Biology, vol 2646. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3060-0_30
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DOI: https://doi.org/10.1007/978-1-0716-3060-0_30
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