Abstract
Objectives
To design a new system for the in vivo phosphorylation of proteins in Escherichia coli using the co-expression of the α-subunit of casein kinase II (CKIIα) and a target protein, (Nanofitin) fused with a phosphorylatable tag.
Results
The level of the co-expressed CKIIα was controlled by the arabinose promoter and optimal phosphorylation was obtained with 2 % (w/v) arabinose as inductor. The effectiveness of the phosphorylation system was demonstrated by electrophoretic mobility shift assay (NUT-PAGE) and staining with a specific phosphoprotein-staining gel. The resulting phosphorylated tag was also used to purify the phosphoprotein by immobilized metal affinity chromatography, which relies on the specific interaction of phosphate moieties with Fe(III).
Conclusion
The use of a single tag for both the purification and protein array anchoring provides a simple and straightforward system for protein analysis.
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Acknowledgments
M.G. was supported by the “Region Pays de la Loire” within the framework of the Erasmus Mundus Program “NanoFar”.
Supporting information
Supplementary Fig. 1—Sequences of Nanofitins H4P and H4P′.
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Goux, M., Fateh, A., Defontaine, A. et al. In vivo phosphorylation of a peptide tag for protein purification. Biotechnol Lett 38, 767–772 (2016). https://doi.org/10.1007/s10529-016-2040-4
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DOI: https://doi.org/10.1007/s10529-016-2040-4
Keywords
- Casein kinase II
- Nanofitin
- Phosphorylation
- Phosphorylated peptide tag
- Protein purification