Tailor-made ezrin actin binding domain to probe its interaction with actin in-vitro

PLoS One. 2015 Apr 10;10(4):e0123428. doi: 10.1371/journal.pone.0123428. eCollection 2015.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Actins / chemistry*
  • Actins / genetics
  • Actins / metabolism*
  • Animals
  • Avian Proteins / chemistry
  • Avian Proteins / genetics
  • Avian Proteins / metabolism
  • Chickens
  • Cytoskeletal Proteins / chemistry*
  • Cytoskeletal Proteins / genetics
  • Cytoskeletal Proteins / metabolism*
  • In Vitro Techniques
  • Microfilament Proteins / chemistry
  • Microfilament Proteins / genetics
  • Microfilament Proteins / metabolism
  • Models, Molecular
  • Protein Interaction Domains and Motifs
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism

Substances

  • Actins
  • Avian Proteins
  • Cytoskeletal Proteins
  • Microfilament Proteins
  • Recombinant Fusion Proteins
  • ezrin

Grant support

This study was supported by Indo Finland and TIFR, Department of Biotechnology, India.