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Tailor-made Ezrin actin binding domain to probe its interaction with actin in-vitro
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Shrivastava, Rohini, Köster, Darius, Kalme, Sheetal, Mayor, Satyajit and Neerathilingam, Muniasamy (2015) Tailor-made Ezrin actin binding domain to probe its interaction with actin in-vitro. PLoS One, 10 (4). e0123428. doi:10.1371/journal.pone.0123428 ISSN 1932-6203.
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Official URL: http://dx.doi.org/10.1371/journal.pone.0123428
Abstract
Ezrin, a member of the ERM (Ezrin/Radixin/Moesin) protein family, is an Actin-plasma membrane linker protein mediating cellular integrity and function. In-vivo study of such interactions is a complex task due to the presence of a large number of endogenous binding partners for both Ezrin and Actin. Further, C-terminal actin binding capacity of the full length Ezrin is naturally shielded by its N-terminal, and only rendered active in the presence of Phosphatidylinositol bisphosphate (PIP2) or phosphorylation at the C-terminal threonine. Here, we demonstrate a strategy for the design, expression and purification of constructs, combining the Ezrin C-terminal actin binding domain, with functional elements such as fusion tags and fluorescence tags to facilitate purification and fluorescence microscopy based studies. For the first time, internal His tag was employed for purification of Ezrin actin binding domain based on in-silico modeling. The functionality (Ezrin-actin interaction) of these constructs was successfully demonstrated by using Total Internal Reflection Fluorescence Microscopy. This design can be extended to other members of the ERM family as well.
Item Type: | Journal Article | ||||
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Divisions: | Faculty of Science, Engineering and Medicine > Medicine > Warwick Medical School > Biomedical Sciences > Cell & Developmental Biology Faculty of Science, Engineering and Medicine > Medicine > Warwick Medical School |
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Journal or Publication Title: | PLoS One | ||||
Publisher: | Public Library of Science | ||||
ISSN: | 1932-6203 | ||||
Official Date: | 10 April 2015 | ||||
Dates: |
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Volume: | 10 | ||||
Number: | 4 | ||||
Article Number: | e0123428 | ||||
DOI: | 10.1371/journal.pone.0123428 | ||||
Status: | Peer Reviewed | ||||
Publication Status: | Published | ||||
Access rights to Published version: | Open Access (Creative Commons) |
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