| 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.
Published: October 25, 2004. |
