Akizuki Kazutoshi, Toyama Taku, Yamashita Masashi, Sugiyama Yasunori, Ishida Atsuhiko, Kameshita Isamu, Sueyoshi Noriyuki
Department of Life Sciences, Faculty of Agriculture, Kagawa University, Kagawa 761-0795, Japan; Research Fellow of Japan Society for the Promotion of Science, Japan.
Department of Life Sciences, Faculty of Agriculture, Kagawa University, Kagawa 761-0795, Japan.
Anal Biochem. 2018 May 15;549:99-106. doi: 10.1016/j.ab.2018.03.015. Epub 2018 Mar 17.
Casein kinase 1 (CK1) is a widely expressed Ser/Thr kinase in eukaryotic organisms that is involved in various cellular processes (e.g., circadian rhythm and apoptosis). Therefore, preparing highly active CK1 and investigating its properties in vitro have important implications for understanding the biological roles of the kinase. However, recombinant CK1 undergoes autoinactivation via autophosphorylation in Escherichia coli cells and thus is undesirably prepared as a phosphorylated and inactivated kinase. To circumvent this problem, we established a protein expression system using E. coli strain BL21(DE3)pλPP in which λ protein phosphatase (λPPase) is constitutively expressed. Using this system, recombinant CK1 isoforms (α, δ and ε) were readily prepared as unphosphorylated forms. Furthermore, we found that CK1s prepared using BL21(DE3)pλPP showed markedly higher activity than those prepared by the conventional BL21(DE3). Finally, we demonstrated that the kinase activity of CK1δ from BL21(DE3)pλPP was higher than that prepared by a conventional method consisting of troublesome steps such as in vitro λPPase treatment. Thus, this simple method using BL21(DE3)pλPP is valuable for preparing highly active CK1s. It may also be applicable to other kinases that are difficult to prepare because of phosphorylation in E. coli cells.
酪蛋白激酶1（CK1）是一种在真核生物中广泛表达的丝氨酸/苏氨酸激酶，参与多种细胞过程（如昼夜节律和细胞凋亡）。因此，制备高活性的CK1并在体外研究其性质对于理解该激酶的生物学作用具有重要意义。然而，重组CK1在大肠杆菌细胞中会通过自磷酸化发生自失活，因此制备得到的是磷酸化且失活的激酶，这并不理想。为了解决这个问题，我们建立了一种使用大肠杆菌BL21(DE3)pλPP菌株的蛋白质表达系统，其中λ蛋白磷酸酶（λPPase）是组成型表达的。利用该系统，重组CK1亚型（α、δ和ε）很容易以未磷酸化的形式制备出来。此外，我们发现使用BL21(DE3)pλPP制备的CK1s比使用传统的BL21(DE3)制备的CK1s具有明显更高的活性。最后，我们证明了来自BL21(DE3)pλPP的CK1δ的激酶活性高于通过包括体外λPPase处理等繁琐步骤的传统方法制备的CK1δ。因此，这种使用BL21(DE3)pλPP的简单方法对于制备高活性的CK1s很有价值。它也可能适用于因在大肠杆菌细胞中发生磷酸化而难以制备的其他激酶。
