title: In vivo phosphorylation of a peptide tag for protein purification.
abstract: OBJECTIVE
  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).
  
  
  CONCLUSIONS
  The use of a single tag for both the purification and protein array anchoring provides a simple and straightforward system for protein analysis.
authors.affiliation: UFIP, UMR 6286 CNRS, Université de Nantes, 2, rue de la Houssinière, 44322, Nantes Cedex 03, France.
authors.forename: Marine
authors.initials: M
authors.lastname: Goux
authors.affiliation: UFIP, UMR 6286 CNRS, Université de Nantes, 2, rue de la Houssinière, 44322, Nantes Cedex 03, France.
authors.forename: Amina
authors.initials: A
authors.lastname: Fateh
authors.affiliation: UFIP, UMR 6286 CNRS, Université de Nantes, 2, rue de la Houssinière, 44322, Nantes Cedex 03, France.
authors.forename: Alain
authors.initials: A
authors.lastname: Defontaine
authors.affiliation: Affilogic SAS, 2, rue de la Houssinière, 44300, Nantes, France.
authors.forename: Mathieu
authors.initials: M
authors.lastname: Cinier
authors.affiliation: UFIP, UMR 6286 CNRS, Université de Nantes, 2, rue de la Houssinière, 44322, Nantes Cedex 03, France. charles.tellier@univ-nantes.fr.
authors.forename: Charles
authors.initials: C
authors.lastname: Tellier
mesh_terms: D047390:Casein Kinase II / Q000235:genetics / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D011506:Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification* / Q000378:metabolism*
chemical_list: D021122:Protein Subunits; D011506:Proteins; D011993:Recombinant Fusion Proteins; D047390:Casein Kinase II
keywords: Casein kinase II; Nanofitin; Phosphorylated peptide tag; Phosphorylation; Protein purification