Purification and ATPase Activity Measurement of Spiroplasma MreB

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 superfam...

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Bibliographic Details
Published inMethods in molecular biology (Clifton, N.J.) Vol. 2646; p. 359
Main Authors Takahashi, Daichi, Fujiwara, Ikuko, Miyata, Makoto
Format Journal Article
LanguageEnglish
Published United States 2023
Subjects
Actins - metabolism
Adenosine Triphosphatases - metabolism
Bacterial Proteins - metabolism
Spiroplasma - metabolism
Absorption spectroscopy
Bacterial actin cytoskeleton
E. coli expression system
Gel filtration
Pi release assay
Ni2+-NTA affinity chromatography
Recombinant protein
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Summary: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 P 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.
ISSN:1940-6029
DOI:10.1007/978-1-0716-3060-0_30