title: Non-covalent modification of granulocyte-colony stimulating factor (G-CSF) by coiled-coil technology.
abstract: 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 5kDa 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.
authors.affiliation: School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Geneva, Switzerland. Electronic address: christian.reichert@hotmail.de.
authors.forename: Christian
authors.initials: C
authors.lastname: Reichert
authors.affiliation: School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Geneva, Switzerland.
authors.forename: Remo
authors.initials: R
authors.lastname: Perozzo
authors.affiliation: School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Geneva, Switzerland.
authors.forename: Gerrit
authors.initials: G
authors.lastname: Borchard
mesh_terms: D002151:Calorimetry / Q000379:methods*; D002942:Circular Dichroism / Q000379:methods*; D016179:Granulocyte Colony-Stimulating Factor / Q000138:chemical synthesis*; D006801:Humans; D011092:Polyethylene Glycols / Q000138:chemical synthesis; D015202:Protein Engineering / Q000379:methods*; D017433:Protein Structure, Secondary
chemical_list: D016179:Granulocyte Colony-Stimulating Factor; D011092:Polyethylene Glycols
keywords: Coiled-coil; G-CSF; Leucine zipper; Non-covalent PEGylation; Protein engineering; Protein modification