Induced heterodimerization and purification of two target proteins by a synthetic coiled-coil tag

Protein Sci. 2012 Apr;21(4):511-9. doi: 10.1002/pro.2035. Epub 2012 Feb 23.


A synthetic de novo designed heterodimeric coiled-coil was used to copurify two target fluorescent proteins, Venus and enhanced cyan fluorescent protein (ECFP). The coiled-coil consists of two 21-amino acid repetitive sequences, (EIAALEK)(3) and (KIAALKE)(3), named E3 and K3, respectively. These sequences were fused to the C-termini of ECFP or Venus followed by either a strep- or a his-tag, respectively, for affinity purification. Mixed lysates of Venus-K3 and ECFP-E3 were subjected to consecutive affinity purification and showed highly specific association between the coiled-coil pair by SDS-PAGE, gel filtration, isothermal titration calorimetry (ITC), and fluorescence resonance energy transfer (FRET). The tagged proteins eluted as heterodimers at the concentrations tested. FRET analysis further showed that the coiled-coil pair was stable in buffers commonly used for protein purification, including those containing high salt concentration and detergent. This study shows that the E3/K3 pair is very well suited for the copurification of two target proteins expressed in vivo because of its high specificity: it forms exclusively heterodimers in solution, it does not interact with any cellular proteins and it is stable under different buffer conditions.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Affinity Labels / chemistry
  • Amino Acid Motifs
  • Amino Acid Sequence
  • Buffers
  • Calorimetry / methods
  • Chromatography, Affinity
  • Cloning, Molecular
  • Electrophoresis, Polyacrylamide Gel
  • Escherichia coli / chemistry
  • Fluorescence Resonance Energy Transfer
  • Green Fluorescent Proteins / chemistry
  • Green Fluorescent Proteins / isolation & purification*
  • Molecular Sequence Data
  • Plasmids / chemistry
  • Protein Denaturation
  • Protein Interaction Mapping
  • Protein Multimerization*
  • Protein Stability
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / isolation & purification*
  • Repetitive Sequences, Amino Acid
  • Salts / chemistry
  • Solutions / chemistry
  • Substrate Specificity


  • Affinity Labels
  • Buffers
  • Recombinant Fusion Proteins
  • Salts
  • Solutions
  • enhanced cyan fluorescent protein
  • Green Fluorescent Proteins