title: Induced heterodimerization and purification of two target proteins by a synthetic coiled-coil tag.
abstract: 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.
authors.affiliation: Research Institute of Molecular Pathology, Dr Bohr Gasse 7, A-1030 Vienna, Austria.
authors.forename: Jesus
authors.initials: J
authors.lastname: Fernandez-Rodriguez
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Marlovits
mesh_terms: D000345:Affinity Labels / Q000737:chemistry; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D002021:Buffers; D002151:Calorimetry / Q000379:methods; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D010957:Plasmids / Q000737:chemistry; D011489:Protein Denaturation; D025941:Protein Interaction Mapping; D055503:Protein Multimerization; D055550:Protein Stability; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification*; D020449:Repetitive Sequences, Amino Acid; D012492:Salts / Q000737:chemistry; D012996:Solutions / Q000737:chemistry; D013379:Substrate Specificity
chemical_list: D000345:Affinity Labels; D002021:Buffers; D011993:Recombinant Fusion Proteins; D012492:Salts; D012996:Solutions; C527701:enhanced cyan fluorescent protein; D049452:Green Fluorescent Proteins