title: Directed immobilization of peptide ligands to accessible pore sites by conjugation with a placeholder molecule.
abstract: When small ligands are immobilized onto a porous chromatography medium, only a limited number of binding sites contributes to the interaction with the target molecule. The main part of the ligand molecules is distributed on sites that are not accessible for the target protein due to steric hindrance. To direct the ligand into a well-accessible position, the ligand was conjugated to a large molecule that acted as a placeholder during the immobilization step. Then the placeholder molecule was cleaved off and washed out. Two linear peptides with affinity for lysozyme and human blood coagulation factor VIII, respectively, were studied as model systems. The protected peptide ligand was covalently linked to a 20-kDa poly(ethylene glycol) molecule containing an acid-labile linker. After selective deprotection of the peptide and purification, immobilization of this conjugate on a preactivated chromatography matrix was performed alternatively through the free N-terminus, the epsilon-amino group of lysine, or the sulfohydryl group of cysteine. After the immobilization reaction, the spacer molecule and remaining protecting groups were cleaved off and the gels were tested by affinity chromatography. This novel immobilization technique substantially increased the binding capacity and the ligand utilization for the target protein, and site-specific immobilization could be demonstrated.
authors.affiliation: Institute for Applied Microbiology, University of Agricultural Sciences, A-1190 Vienna, Austria.
authors.forename: Rainer
authors.initials: R
authors.lastname: Hahn
authors.forename: Eva
authors.initials: E
authors.lastname: Berger
authors.forename: Karin
authors.initials: K
authors.lastname: Pflegerl
authors.forename: Alois
authors.initials: A
authors.lastname: Jungbauer
mesh_terms: D002846:Chromatography, Affinity / Q000379:methods*; D020650:Combinatorial Chemistry Techniques; D003432:Cross-Linking Reagents; D008024:Ligands; D019151:Peptide Library; D010455:Peptides / Q000138:chemical synthesis*; D016062:Porosity
chemical_list: D003432:Cross-Linking Reagents; D008024:Ligands; D019151:Peptide Library; D010455:Peptides