Kinase-Glo激酶发光法体外测定蛋白激酶A活性

Kinase-Glo luminescent kinase assay for in vitro determination of PKA activity

目的:建立一种简便、无害的体外测定PKA激酶活性的非放射性核素方法。方法:采用RT-PCR方法从HEK293细胞的总RNA中扩增PKAα的cDNA,并将PKAα的cDNA克隆至pGEX-6p-1载体中。经纯化后的体外表达蛋白用免疫印迹(Western blot)法进行鉴定。最后采用非放射性核素法Kinase-Glo激酶发光检测试剂盒测定表达蛋白GST-PKAα的活性。结果:经过优化诱导条件,我们成功地纯化得到了GST-PKAα的融合蛋白。用非放射性核素法Kinase-Glo激酶发光检测试剂盒测定纯化蛋白GST-PKAα活性。我们还用PKA特异性抑制剂H-89进一步确定了表达蛋白GST-PKAα的活性,并且测定了其IC50值为(35.2±3.97)nmol/L,与报道值一致。结论:Kinase-Glo激酶发光检测法测定PKA激酶活性,操作简便、对人体无害。此方法能有效应用于临床前PKA激酶抑制剂的高通量筛选、PKA激酶新作用靶点的发现以及靶向蛋白PKA磷酸化位点的研究。

AIM： To establish a simple, convenient and harmless non-radioactive method of determining protein kinase A （PKA） activity in vitro. METHODS： Human PKAα cDNA was amplified from total RNA of HEK293 cells using RT-PCR method and then cloned into pGEX-6p-1 vector. In vitro purified GST-PKAα protein was identified by Western blot analysis. Finally, a non-radioactive method, Kinase-GIo luminescent kinase assay, was employed to determine the kinase activity of purified GST-PKAcx. RESULTS： After the optimization of the induction conditions, we purified GST- PKAα protein successfully. We then determined GST-PKAα activity using Kinase-GIo luminescent kinase assay. We also further confirmed the kinase activity of GST-PKAcc using H-89, a specific PKA inhibitor, and determined its ICsovalue （35.2 ± 3.97 ） nmol/L which is consistent with reported value. CONCLUSION： The non-radioactive Kinase-GIo luminescent kinase assay is a simple, convenient and harmless method of determining the kinase activity of PKA. This method is effective for pre-clinical high-throughput screening of PKA inhibitor, discovering novel target proteins of PKA and investigating PKA phosphorylation sites in target proteins.