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

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.