Please use this identifier to cite or link to this item: http://dr.iiserpune.ac.in:8080/xmlui/handle/123456789/9142
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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