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Heidi Rommelaere^1*, Davy Waterschoot¹, Katrien Neirynck¹, Joël Vandekerckhove¹ and Christophe Ampe¹

¹ Flanders Interuniversity Institute for Biotechnology (VIB 09) and Department of Biochemistry, Faculty of Medicine and Health Sciences, Ghent University. B-9000 Gent. Belgium.

* To whom correspondence should be addressed: Heidi Rommelaere, Flanders Interuniversity Institute for Biotechnology (VIB 09) and Department of Biochemistry, Faculty of Medicine and Health Sciences, Ghent University. B-9000 Gent. Belgium. Phone: 32 9 264 93 33. Fax: 32 9 264 94 88. Email: Heidi.Rommelaere@ugent.be

Biol. Proced. Online 2004;6:235-249. doi:10.1251/bpo94
Submitted: February 18, 2004; Accepted: October 01, 2004; Published: October 25, 2004.

Indexing terms: Actins; Binding sites; Protein folding.

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.