Protein detection by Western blot via coiled-coil interactions.
We propose an approach for the detection of proteins by Western blot that takes advantage of the high-affinity interaction occurring between two de novo designed peptides, the E and K coils. As a model system, K coil-tagged epidermal growth factor (EGF) was revealed with secreted alkaline phosphatase (SeAP) tagged with E coil (SeAP-Ecoil) as well as with biotinylated E coil. In that respect, we first produced purified SeAP-Ecoil and verified its ability to interact with K coil peptides by surface plasmon resonance biosensing. We demonstrated that protein detection with Ecoil-biotin was more specific than with SeAP-Ecoil. We then showed that our approach is as sensitive as conventional detection strategies relying on nickel-nitrilotriacetic acid-horseradish peroxidase (Ni-NTA-HRP), anti-His-HRP, or anti-EGF. Altogether, our results indicate that the E/K coiled-coil system is a good alternative for protein detection by Western blot.
Boucher
Cyril
C
Animal Cell Technology Group, Bioprocess Center, Biotechnology Research Institute, National Research Council Canada, Montréal, Quebec H4P 2R2, Canada.
St-Laurent
Gilles
G
Jolicoeur
Mario
M
Crescenzo
Gregory De
GD
Durocher
Yves
Y
D000469:Alkaline Phosphatase / Q000737:chemistry*; D000595:Amino Acid Sequence; D015153:Blotting, Western / Q000379:methods*; D002460:Cell Line; D004815:Epidermal Growth Factor / Q000737:chemistry*; D006801:Humans; D054730:Protein Interaction Domains and Motifs; D017433:Protein Structure, Secondary
D004815:Epidermal Growth Factor; D000469:Alkaline Phosphatase