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In vivo phosphorylation of a peptide tag for protein purification

Biotechnology Letters, ISSN: 1573-6776, Vol: 38, Issue: 5, Page: 767-772
2016
  • 4
    Citations
  • 0
    Usage
  • 16
    Captures
  • 0
    Mentions
  • 0
    Social Media
Metric Options:   Counts1 Year3 Year

Metrics Details

  • Citations
    4
    • Citation Indexes
      4
  • Captures
    16

Article Description

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.

Bibliographic Details

Goux, Marine; Fateh, Amina; Defontaine, Alain; Cinier, Mathieu; Tellier, Charles

Springer Science and Business Media LLC

Biochemistry, Genetics and Molecular Biology; Chemical Engineering; Immunology and Microbiology

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