Oakhill Jonathan S, Scott John W, Dite Toby A
Metabolic Signalling Laboratory, St. Vincent's Institute of Medical Research, Fitzroy, VIC, Australia.
Mary MacKillop Institute for Health Research, Australian Catholic University, Melbourne, VIC, Australia.
Methods Mol Biol. 2018;1732:159-169. doi: 10.1007/978-1-4939-7598-3_10.
Regulation of AMP-activated protein kinase (AMPK) signalling is complex and involves contributions from adenine nucleotides, co-/posttranslational modifications, and isoform composition of the AMPK heterotrimer. It is becoming apparent that AMPK activation/inhibition by synthetic drugs involves similar levels of complexity. Major advances in our understanding of these mechanisms have been gained from recombinant expression systems that provide sufficient quantities of highly purified material for structure/function studies. Here, we provide a detailed protocol for transient expression of affinity-tagged AMPK complexes in mammalian cells. We have found this system to be optimal as a source of enzyme possessing regulatory modifications found in vivo.
AMP 激活的蛋白激酶（AMPK）信号传导的调节是复杂的，涉及腺嘌呤核苷酸、共翻译/翻译后修饰以及 AMPK 异源三聚体的亚型组成。越来越明显的是，合成药物对 AMPK 的激活/抑制也涉及类似程度的复杂性。我们对这些机制的理解取得的主要进展来自重组表达系统，该系统提供了足够数量的高度纯化材料用于结构/功能研究。在这里，我们提供了一个在哺乳动物细胞中瞬时表达亲和标签 AMPK 复合物的详细方案。我们发现这个系统作为具有体内发现的调节修饰的酶的来源是最佳的。
