Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia; Department of Medical Biology, University of Melbourne, Parkville, VIC, Australia.
Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia; Department of Medical Biology, University of Melbourne, Parkville, VIC, Australia.
Methods Enzymol. 2022;667:183-227. doi: 10.1016/bs.mie.2022.03.029. Epub 2022 Apr 8.
Pseudokinase domains are found throughout the kingdoms of life and serve myriad roles in cell signaling. These domains, which resemble protein kinases but are catalytically-deficient, have been described principally as protein interaction domains. Broadly, pseudokinases have been reported to function as: allosteric regulators of conventional enzymes; scaffolds to nucleate assembly and/or localization of signaling complexes; molecular switches; or competitors of signaling complex assembly. From detailed structural and biochemical studies of individual pseudokinases, a picture of how they mediate protein interactions is beginning to emerge. Many such studies have relied on recombinant protein production in insect cells, where endogenous chaperones and modifying enzymes favor bona fide folding of pseudokinases. Here, we describe methods for co-expression of pseudokinases and their interactors in insect cells, as exemplified by the MLKL pseudokinase, which is the terminal effector in the necroptosis cell death pathway, and its upstream regulator kinase RIPK3. These methods are broadly applicable to co-expression of other pseudokinases with their interaction partners from bacmids using the baculovirus-insect cell expression system.
假激酶结构域存在于生命的各个领域，在细胞信号转导中发挥着多种作用。这些结构域类似于蛋白激酶，但缺乏催化活性，主要被描述为蛋白相互作用结构域。广义上，假激酶被认为具有以下功能：传统酶的变构调节剂；组装和/或定位信号复合物的核酶；分子开关；或信号复合物组装的竞争者。通过对单个假激酶的详细结构和生化研究，开始揭示它们介导蛋白相互作用的方式。许多此类研究依赖于昆虫细胞中重组蛋白的生产，其中内源性伴侣蛋白和修饰酶有利于假激酶的真实折叠。在这里，我们描述了在昆虫细胞中共表达假激酶及其相互作用物的方法，以 MLKL 假激酶为例，它是细胞坏死性细胞死亡途径中的末端效应物，以及其上游调节激酶 RIPK3。这些方法广泛适用于使用杆状病毒-昆虫细胞表达系统从 bacmids 中共表达其他假激酶与其相互作用伙伴。
