Maki Jenny L, Tres Brazell J, Teng Xin, Cuny Gregory D, Degterev Alexei
Department of Biochemistry, School of Medicine, Tufts University, Boston, MA 02111, USA.
Protein Expr Purif. 2013 Jun;89(2):156-61. doi: 10.1016/j.pep.2013.03.002. Epub 2013 Mar 21.
Receptor Interacting Protein 1 (RIP1) kinase is one of the key mediators of tumor necrosis factor alpha (TNF-α) signaling and is critical for activation of necroptotic cell death. We developed a method for expression of recombinant kinase, utilizing baculovirus co-infection of Cdc37, an Hsp90 co-chaperone, and RIP1-His, followed by a two-step purification scheme. After optimization, 1-3mg of highly purified RIP1 kinase was typically obtained from a 1L of Sf9 cells. The recombinant protein displayed kinase activity that was blocked by RIP1 inhibitors, necrostatins. The purified protein was used to develop a simple and robust thermal shift assay for further assessment of RIP1 inhibitors.
受体相互作用蛋白1（RIP1）激酶是肿瘤坏死因子α（TNF-α）信号传导的关键介质之一，对坏死性细胞死亡的激活至关重要。我们开发了一种表达重组激酶的方法，利用杆状病毒共感染热休克蛋白90（Hsp90）的共伴侣分子Cdc37和RIP1-组氨酸标签蛋白（RIP1-His），然后采用两步纯化方案。优化后，通常从1升Sf9细胞中获得1-3毫克高度纯化的RIP1激酶。重组蛋白表现出的激酶活性被RIP1抑制剂坏死素所阻断。纯化后的蛋白用于开发一种简单且可靠的热位移分析方法，以进一步评估RIP1抑制剂。
