Soeno Y, Abe H, Kimura S, Maruyama K, Obinata T
Department of Biology, Faculty of Science, Chiba University, Japan.
J Muscle Res Cell Motil. 1998 Aug;19(6):639-46. doi: 10.1023/a:1005329114263.
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.
β -肌动蛋白结合蛋白（CapZ）是一种异二聚体肌动蛋白结合蛋白，它能封闭肌动蛋白丝的带刺末端，并以不依赖Ca2+的方式促进肌动蛋白聚合。在肌原纤维中，它定位于Z线。根据β -肌动蛋白的这些特性，可以推测β -肌动蛋白参与肌动蛋白组装的调节，尤其是在肌原纤维形成过程中I - Z - I复合体的形成。在本研究中，我们设计了一个在大肠杆菌中产生功能性β -肌动蛋白的系统。β -肌动蛋白的βI'和βII亚基的cDNA通过RT - PCR方法获得，以已发表的序列为参考，并亚克隆到pET载体中。当在大肠杆菌中用βI'或βII的cDNA产生蛋白质时，这些蛋白质是不溶性的且无功能。然而，当将编码这两个亚基的cDNA克隆到一个单一载体中并同时表达这两种蛋白质时，这些蛋白质变得可溶，并作为功能性异二聚体被纯化。纯化蛋白质的活性与从骨骼肌中纯化的β -肌动蛋白的活性没有区别。
