Purification and ATPase Activity Measurement of Spiroplasma MreB

Daichi Takahashi; Ikuko Fujiwara; Makoto Miyata

doi:10.1007/978-1-0716-3060-0_30

Purification and ATPase Activity Measurement of Spiroplasma MreB

Methods Mol Biol. 2023:2646:359-371. doi: 10.1007/978-1-0716-3060-0_30.

Authors

Daichi Takahashi^{1

2}, Ikuko Fujiwara^{3

4

5

6}, Makoto Miyata^{1

2

7

8}

Affiliations

¹ Graduate School of Science, Osaka City University, Osaka, Japan.
² Graduate School of Science, Osaka Metropolitan University, Osaka, Japan.
³ Graduate School of Science, Osaka City University, Osaka, Japan. ikukofujiwara@vos.nagaokaut.ac.jp.
⁴ The OCU Advanced Research Institute for Natural Science and Technology (OCARINA), Osaka City University, Osaka, Japan. ikukofujiwara@vos.nagaokaut.ac.jp.
⁵ Department of Bioengineering, Nagaoka University of Technology, Nagaoka, Niigata, Japan. ikukofujiwara@vos.nagaokaut.ac.jp.
⁶ Department of Materials Science and Bioengineering, Nagaoka University of Technology, Nagaoka, Niigata, Japan. ikukofujiwara@vos.nagaokaut.ac.jp.
⁷ The OCU Advanced Research Institute for Natural Science and Technology (OCARINA), Osaka City University, Osaka, Japan.
⁸ The OMU Advanced Research Center for Natural Science and Technology, Osaka Metropolitan University, Osaka, Japan.

PMID: 36842130
DOI: 10.1007/978-1-0716-3060-0_30

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 P_i 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.

Keywords: Absorption spectroscopy; Bacterial actin cytoskeleton; E. coli expression system; Gel filtration; Ni2+-NTA affinity chromatography; Pi release assay; Recombinant protein.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Actins* / metabolism
Adenosine Triphosphatases / metabolism
Bacterial Proteins / metabolism
Spiroplasma* / metabolism

Substances

Actins
Adenosine Triphosphatases
Bacterial Proteins