Co-expression of recombinant RIPK3:MLKL complexes using the baculovirus-insect cell system

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.