Objectives To design a new system for the in vivo phosphorylation of proteins in Escherichia coli using the co-expression of the alpha-subunit of casein kinase II (CKII alpha) and a target protein, (Nanofitin) fused with a phosphorylatable tag. Results The level of the co-expressed CKII alpha 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.