In vivo phosphorylation of a peptide tag for protein purification

• Published in: Biotechnology letters 2016-05, Vol.38 (5), p.767-772
• Main Authors: Goux, Marine, Fateh, Amina, Defontaine, Alain, Cinier, Mathieu, Tellier, Charles
• Format: Article
• Language: English
• Subjects: Applied Microbiology; Arabinose; Arrays; Biochemistry; Biomedical and Life Sciences; Biotechnology; Casein Kinase II - genetics; Casein Kinase II - metabolism; E coli; Electrophoretic Mobility Shift Assay; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Gene Expression; Inductors; Iron; Kinases; Life Sciences; Microbiology; Nanostructure; Original Research Paper; Phosphorylation; Protein Processing, Post-Translational; Protein Subunits - genetics; Protein Subunits - metabolism; Proteins; Proteins - isolation & purification; Proteins - metabolism; Purification; Recombinant Fusion Proteins - isolation & purification; Recombinant Fusion Proteins - metabolism
• ISSN: 0141-5492; 1573-6776
• DOI: 10.1007/s10529-016-2040-4

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.