蛋白激酶AMPK高效制备及活性检测方法的建立

A novel method for the production of protein kinase AMPK and the establishment of its kinase activity assay

目的 建立高效的腺苷酸依赖的蛋白激酶(AMPK)的制备方法,并建立1种基于酶联免疫吸附(ELISA)法的AMPK的激酶活性检测方法以用于药物筛选。方法 利用大肠杆菌表达系统,将AMPK的α、β和γ 3个亚基与钙调素依赖性蛋白激酶β(CAMKK β)共表达,重组AMPK被CAMKK β磷酸化修饰并激活,进而纯化得到具有激酶活性的重组AMPK蛋白复合体。根据AMPK底物乙酰辅酶A羧化酶(ACC)的磷酸化修饰位点(Ser79)设计合成生物素标记的多肽(SAMS)作为AMPK激酶的底物。在ATP存在的情况下AMPK对生物素标记的SAMS多肽进行磷酸化修饰,反应产物转移到链霉亲和素包被的酶标板中,SAMS多肽通过链霉亲和素结合到酶标板。以能够识别SAMS磷酸化修饰的抗体作为一抗进行ELISA检测,SAMS的磷酸化修饰水平反应AMPK的激酶活性。结果 利用大肠杆菌表达系统成功得到有激酶活性的AMPK蛋白复合体,建立了ELISA检测AMPK的激酶活性方法。通过对AMPK激活剂A-769662检测,证明该方法可以用于AMPK激活剂/抑制剂的筛选。结论 通过构建大肠杆菌共表达系统,成功获得了AMPK激酶,与传统方法相比更为简洁高效;建立的AMPK的激酶活性检测方法操作简单、准确度高且安全高效,有望用于海洋活性分子的大规模筛选,加快我国“蓝色药库”的开发。

Objective To establish an efficient production method for AMP-dependent protein kinase(AMPK), and to establish a method for detecting AMPK protein kinase activity based on enzyme-linked immunosorbent assay(ELISA). Methods Using the E. coli expression system, the three subunits of AMPK, α, β, and γ, were co-expressed with calmodulin-dependent protein kinase kinase β(CAMKK β). Recombinant AMPK proteins were phosphorylated by CAMKK β, and then purified. Based on the phosphorylation site(Ser79) of acetyl-Coenzyme A carboxylase(ACC), a biotin-labeled peptide(SAMS) was designed and synthesized as AMPK kinase substrate. In the presence of ATP and SAMS, the kinase assay of AMPK was carried out, and the reaction product was transferred to the streptavidin-coated ELISA plate, and the biotin-labeled SAMS polypeptides were bound the ELISA plate. An antibody capable of recognizing the phosphorylation of SAMS was used as the primary antibody for ELISA detection, and the level of phosphorylation of SAMS could reflect the kinase activity of AMPK. Results In this paper, an AMPK protein complex with kinase activity was successfully obtained using the E. coli expression system, and an ELISA method for detecting AMPK protein kinase activity was established. By testing the commercial AMPK activator, A-769662, it was proved that this method could be used for AMPK activator/inhibitor screening. Conclusion In this paper, by constructing an E. coli co-expression system, AMPK kinase was successfully obtained, which was more concise and efficient than traditional methods;the established AMPK kinase assay method was simple operation, high accuracy, safety and efficiency, and was expected to be used in the high-throughput screening of marine natural products, which would accelerate the development of the marine drugs in China.