Generation of functional β-actinin (CapZ) in an E-coli expression system

beta-actinin (CapZ) is a heterodimeric actin-binding protein which caps the barbed end of actin filaments and nucleates actin-polymerization in a Ca2+-independent manner. In myofibrils it is localized in the Z-lines. As judged by these properties of beta-actinin, it is conceivable that beta-actinin is involved in the regulation of actin assembly, especially in the formation of I-Z-I complex during myofibrillogenesis. In this study, we devised a system to produce functional beta-actinin in E. Coli. The cDNAs of beta I' and beta II subunits of beta-actinin were obtained by RT-PCR methods using the published sequence as references, and subcloned in a pET vector. When the proteins were produced with the cDNA of either beta I' or beta II in E. coli, the proteins were insoluble and non-functional. However, when the cDNAs encoding the two subunits were cloned into a single vector and both proteins were expressed simultaneously, the proteins became soluble and purified as a functional heterodimer. The activity of the purified proteins was not distinguishable from that of beta-actinin purified from skeletal muscle. (C) Chapman & Hall Ltd.