Generation of functional beta-actinin (CapZ) in an E. coli expression system.
beta-actinin (CapZ) is a heterodimeric actin-binding protein which caps the barbed end of action 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 b-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 myofribrillogenesis. 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' and 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.
Soeno
Y
Y
Department of Biology, Faculty of Science, Chiba University, Japan.
Abe
H
H
Kimura
S
S
Maruyama
K
K
Obinata
T
T
D000185:Actinin / Q000096:biosynthesis* / Q000235:genetics* / Q000502:physiology; D000199:Actins / Q000378:metabolism; D000818:Animals; D051347:CapZ Actin Capping Protein; D002642:Chick Embryo; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors / Q000378:metabolism*; D008840:Microfilament Proteins; D009124:Muscle Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000502:physiology; D009210:Myofibrils / Q000737:chemistry; D011485:Protein Binding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism
D000199:Actins; D051347:CapZ Actin Capping Protein; D008840:Microfilament Proteins; D009124:Muscle Proteins; D011994:Recombinant Proteins; D000185:Actinin