Open Access

A method for rapidly screening functionality of actin mutants and tagged actins

  • Heidi Rommelaere1Email author,
  • Davy Waterschoot1,
  • Katrien Neirynck1,
  • Joël Vandekerckhove1 and
  • Christophe Ampe1
Biological Procedures Online6:610235

https://doi.org/10.1251/bpo94

Received: 18 February 2004

Accepted: 1 October 2004

Abstract

Recombinant production and biochemical analysis of actin mutants has been hampered by the fact that actin has an absolute requirement for the eukaryotic chaperone CCT to reach its native state. We therefore have developed a method to rapidly screen the folding capacity and functionality of actin variants, by combining in vitro expression of labelled actin with analysis on native gels, band shift assays or copolymerization tests. Additionally, we monitor, using immuno-fluorescence, incorporation of actin variants in cytoskeletal structures in transfected cells. We illustrate the method by two examples. In one we show that tagged versions of actin do not always behave native-like and in the other we study some of the molecular defects of three β-actin mutants that have been associated with diseases.

Indexing terms

Actins Binding sites Protein folding

Notes

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