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Title: | Improving Stability of Spiroplasma citri MreB5 Through Purification Optimization and Structural Insights |
Authors: | PANDE, VANI PANANGHAT, GAYATHRI, Dept. of Biology |
Keywords: | MreB Cytoskeleton proteins ADP Thermal shift assay Affinity chromatography Size exclusion chromatography Crystallization X-Ray adsorption spectroscopy 2024 2024-OCT-WEEK2 TOC-OCT-2024 |
Issue Date: | Oct-2024 |
Publisher: | Bio-protocol LLC |
Citation: | Bio-protocol, 14(20). |
Abstract: | MreB is a prokaryotic actin homolog. It is essential for cell shape in the majority of rod-shaped cell-walled bacteria. Structural and functional characterization of MreB protein is important to understand the mechanism of ATP-dependent filament dynamics and membrane interaction. In vitro studies on MreBs have been limited due to the difficulty in purifying the homogenous monomeric protein. We have purified MreB from the cell-wall-less bacteria Spiroplasma citri, ScMreB5, using heterologous expression in Escherichia coli. This protocol provides a detailed description of purification condition optimization that led us to obtain high concentrations of stable ScMreB5. Additionally, we have provided a protocol for detecting the presence of monovalent ions in the ScMreB5 AMP-PNP-bound crystal structure. This protocol can be used to obtain a high yield of ScMreB5 for carrying out biochemical and reconstitution studies. The strategies used for ScMreB5 show how optimizing buffer components can enhance the yield and stability of purified protein. |
URI: | https://doi.org/10.21769/BioProtoc.5086 http://dr.iiserpune.ac.in:8080/xmlui/handle/123456789/9142 |
ISSN: | 2331-8325 |
Appears in Collections: | JOURNAL ARTICLES |
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