A method for rapidly screening functionality of actin mutants and tagged actins.
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 beta-actin mutants that have been associated with diseases.
Rommelaere
Heidi
H
Flanders Interuniversity Institute for Biotechnology (VIB 09) and Department of Biochemistry, Faculty of Medicine and Health Sciences, Ghent University. B-9000 Gent. Belgium.
Waterschoot
Davy
D
Neirynck
Katrien
K
Vandekerckhove
Joël
J
Ampe
Christophe
C