We present here an approach to non-covalently combine an engineered model protein with a PEGylated peptide via coiled-coil binding. To this end a fusion protein of G-CSF and the peptide sequence (JunB) was created-one sequence of JunB was expressed at the N-terminal of GCSF. JunB is able to bind to the peptide sequence cFos, which was in turn covalently linked to a chain of poly(ethylene glycol) (PEG). The selected peptide sequences are leucine zipper motives from transcription factors and are known to bind to each other specifically by formation of a super secondary structure called coiled-coil. The binding between PEGylated peptides of various molecular weights and the modified protein was assessed by isothermal calorimetry (ITC), dynamic light scattering (DLS), circular dichroism (CD), and fluorescence anisotropy.
Our findings show that the attachment of 2 and 5 kDa PEG does not interfere with coiled-coil formation and thus binding of peptide to fusion protein. With this work we successfully demonstrate the non-covalent binding of a model moiety (PEG) to a protein through coiled-coil interaction.