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  • In this study CK C prepared from BL DE p

    2019-12-14

    In this study, CK1δ(ΔC) prepared from BL21(DE3)pλPP had the highest kinase activity among the three isoforms tested when using casein as a substrate. Therefore, CK1δ(ΔC) from BL21(DE3)pλPP has the potential to be a “phosphorylating reagent” that may be widely used in experiments of phosphorylation and dephosphorylation of acidic proteins, because CK1s are also known to preferably phosphorylate acidic substrates [33] (e.g., caseins and D4 peptide [34]). We have previously reported that the catalytic subunit of cAMP-dependent protein kinase (PKAc) and the C-terminal Isoprenaline HCl mutant of zebrafish Ca2+/calmodulin-dependent protein kinase Iδ [zCaMKIδ(1–299)] can efficiently phosphorylate acidic/neutral and basic proteins, respectively [35,36]. Given this information, CK1δ(ΔC) prepared from BL21(DE3)pλPP could be an alternative and complement to PKAc and zCaMKIδ(1–299). The novel E. coli strain in which λPPase is constitutively expressed, designated here as BL21(DE3)pλPP, is actually applicable not only for CK1s but also for other PKs (Fig. S2). Besides CK1s, a number of PKs undergo autophosphorylation in E. coli cells, and thus are often prepared as unexpectedly phosphorylated forms [24,37,38]. Because autophosphorylation can largely alter their biochemical properties including kinase activity or binding to their modulators, use of the undesirably autophosphorylated kinase preparations may lead to misunderstanding of bona fide properties of PKs similarly to CK1s. Furthermore, some proteins other than PKs expressed in E. coli may be unexpectedly phosphorylated by endogenous E. coli PKs [39]. Nevertheless, many studies on recombinant PKs and other phosphorylatable proteins have been thus far performed without considering (auto)phosphorylation in E. coli cells. Notably, this method is more efficient and generally applicable than the previous method as commonly used expression plasmids such as pET vectors harboring each PK gene can be directly used to transform BL21(DE3)pλPP instead of the conventional BL21(DE3). In contrast, the previous methods need to reconstruct the dedicated expression plasmid from the conventional expression vectors for each PK before transformation [22]. Using the BL21(DE3)pλPP expression system reported here, we can now more readily prepare PKs as “naked” forms, free of undesirable phosphorylations (Fig. 7). Reinvestigation of kinases using this expression Isoprenaline HCl method may uncover hidden properties, which have been overlooked so far due to unexpected phosphorylation.
    Acknowledgements This work was supported by JSPS KAKENHI Grant Numbers JP15K07842 to N.S. and JP17J09227 to K.A.