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  • It would be very useful if unphosphorylated

    2020-03-16

    It would be very useful if unphosphorylated CK1s could be prepared using an E. coli expression system without the troublesome in vitro phosphatase treatment. So far, it has been shown that coexpressed λPPase, a Mn2+-dependent protein phosphatase that can dephosphorylate phospho-Ser/Thr/Tyr [20,21], can suppress the autophosphorylation of overexpressed kinases, such as double-strand RNA-activated protein kinase [22], eukaryotic dacomitinib receptor factor 2 kinase [23] and mitogen-activated protein kinases [24], in E. coli cells. In this study, we improved these systems to develop a simple and more efficient method for preparing unphosphorylated protein kinases (PKs) from E. coli cells. We established the novel E. coli strain, BL21(DE3)pλPP, in which λPPase is constitutively expressed, and applied this system to obtain highly active CK1s. Here, we selected three CK1 isoforms, α, δ and ε, which are known to be particularly important in various cellular processes and have been well studied regarding the role of autophosphorylation, and used the novel strain to prepare these isoforms. All the CK1 isoforms were readily prepared by a simple protocol as highly active forms, because they were almost completely dephosphorylated by the coexpressed λPPase in E. coli. Moreover, the truncated forms, CK1δ(ΔC) and CK1ε(ΔC), were prepared as more active forms than those prepared by conventional BL21(DE3) cells. These results suggest that the use of CK1s and their truncated forms obtained from BL21(DE3)pλPP is favorable for the evaluation of the bona fide activity of CK1s in in vitro analyses. We also compared the kinase activity of CK1δ and CK1δ(ΔC) prepared using BL21(DE3)pλPP with the activity of those prepared by a conventional method in which CK1s were dephosphorylated by λPPase in vitro. These experiments demonstrated that the former had higher specific activity than the latter, indicating that the simple method using BL21(DE3)pλPP is useful for preparation of highly active, unphosphorylated CK1s. Application of this method for preparation of other kinases or proteins is also discussed.
    Materials and methods
    Results
    Discussion In the present study, we developed a new protein expression system using BL21(DE3)pλPP, and demonstrated that recombinant CK1s (α, δ and ε) can be readily prepared as unphosphorylated and highly active forms just using this system. Furthermore, CK1δ(ΔC) and CK1ε(ΔC), which are widely used as constitutively active forms, from BL21(DE3)pλPP also exhibited higher kinase activity, compared with those from conventional BL21(DE3) (Fig. 5). Additionally, this method can be used for preparing other CK1 isoforms because CK1β (a CK1 isoform identified in cows [4]) is very similar to the α isoform [9] and autophosphorylation of CKi3 (a CK1γ homolog in Schizosaccharomyces pombe) causes inhibition of the kinase activity [12]. Thus, BL21(DE3)pλPP may be a useful tool for preparing both full-length and C-terminally truncated CK1s as highly active kinases. We directly compared the method using BL21(DE3)pλPP with the conventional method in which CK1s are dephosphorylated by λPPase in vitro and used for kinase assays (Fig. 6), and found that special care must be taken with the latter method for the following reasons: (1) An unexpectedly large amount of λPPase (1 μg) is necessary for complete dephosphorylation of recombinant CK1δ (100 ng). (2) The effect of excessive λPPase (1 μg) is not completely overridden by addition of 1 mM Na3VO4 (data not shown). (3) Na3VO4 (higher than 5 mM) inhibits the kinase activity of CK1δ. Thus, the in vitro PPase treatment required for the conventional method is somewhat tedious and it is difficult to control the reaction. Moreover, the commercially available PPases used for this treatment are relatively expensive. In contrast, unphosphorylated PKs can be readily produced by expressing them in BL21(DE3)pλPP, followed by a simple one-step affinity chromatography, through which we can obtain the purified kinase with essentially no significant contamination of coexpressed λPPase (Supplementary Fig. S1). Considering these results, the BL21(DE3)pλPP method is easier to use and is a low-cost method for preparing fully active CK1s.