Gao Peng, Kolenovsky Allan, Cui Yuhai, Cutler Adrian J, Tsang Edward W T
Plant Biotechnology Institute, National Research Council of Canada, Saskatoon, Saskatchewan, Canada S7N 0W9.
Protein Expr Purif. 2012 Nov;86(1):45-52. doi: 10.1016/j.pep.2012.08.013. Epub 2012 Sep 14.
CIPK3 is a member of CBL (calcineurin B-like)-interacting serine-threonine protein kinases which play an important role in many developmental and adaptation processes in Arabidopsis. Studies conducted on members of this family such as SOS2, PKS8 and PKS11 have provided insight into how these kinases interact with their target substrates in the signal-response process. Since SOS2, PKS8 and PKS11 have low enzymatic activities in vitro and their amino acid sequences are homologous to that of CIPK3, it was assumed that CIPK3 would have a low enzymatic activity. To enhance CIPK3 enzyme activity, a constitutively active form, CIPK3T183D, was generated by a Thr(183) to Asp(183) substitution in the activation loop. To obtain proteins for analysis, glutathione S-transferase (GST) fusion protein system was used. Although both CIPK3 and CIPK3T183D were successfully expressed, they were found in inclusion bodies with three truncated proteins. Since the truncated proteins had a similar affinity to the GST-Bind Resin as the target protein, the one-step affinity purification could no longer be used. As an alternative, His fusion protein expression system was employed for protein production. Although both His-CIPK3 and His-CIPK3T183D also accumulated in inclusion bodies, they were expressed as a single protein species. A method involving Sarkosyl was developed for isolating and purifying the His fusion proteins. His-CIPK3 and His-CIPK3T183D produced were highly purified and enzymatically active. In addition, a 9-fold increase in kinase activity in His-CIPK3T183D was observed, indicating that Thr(183) to Asp(183) substitution in the activation loop of CIPK3 had succeeded in enhancing the kinase activity.
CIPK3是与类钙调神经磷酸酶B（CBL）相互作用的丝氨酸-苏氨酸蛋白激酶家族成员，该家族在拟南芥的许多发育和适应过程中发挥重要作用。对该家族成员如SOS2、PKS8和PKS11的研究，为了解这些激酶在信号响应过程中如何与靶底物相互作用提供了线索。由于SOS2、PKS8和PKS11在体外具有较低的酶活性，且它们的氨基酸序列与CIPK3同源，因此推测CIPK3也具有较低的酶活性。为提高CIPK3的酶活性，通过将激活环中的苏氨酸（Thr183）替换为天冬氨酸（Asp183），构建了组成型激活形式CIPK3T183D。为获得用于分析的蛋白质，使用了谷胱甘肽S-转移酶（GST）融合蛋白系统。虽然CIPK3和CIPK3T183D均成功表达，但它们与三种截短蛋白一起存在于包涵体中。由于截短蛋白与靶蛋白对GST结合树脂具有相似的亲和力，因此无法再使用一步亲和纯化法。作为替代方法，采用His融合蛋白表达系统来生产蛋白质。虽然His-CIPK3和His-CIPK3T183D也在包涵体中积累，但它们以单一蛋白形式表达。开发了一种涉及十二烷基肌氨酸钠的方法来分离和纯化His融合蛋白。所产生的His-CIPK3和His-CIPK3T183D得到了高度纯化且具有酶活性。此外，观察到His-CIPK3T183D的激酶活性增加了9倍，表明CIPK3激活环中苏氨酸（Thr183）到天冬氨酸（Asp183）的替换成功提高了激酶活性。
