title: A biomimetic Protein G affinity adsorbent: an Ugi ligand for immunoglobulins and Fab fragments based on the third IgG-binding domain of Protein G.
abstract: This work reports the development of a synthetic affinity adsorbent for immunoglobulins based on the Fab-binding domain of Streptococcal Protein G (SpG-domain III). The ligand (A2C7I1) was synthesized by the four-component Ugi reaction to generate a substituted peptoidal scaffold mimicking key amino acid residues of SpG. Computer-aided analysis suggests a putative binding site on the CH 1 domain of the Fab molecule. In silico studies, supported by affinity chromatography in comparison with immobilized SpG, as well as analytical characterization by liquid chromatography/electrospray ionization-mass spectrometry and (1) H nuclear magnetic resonance of the ligand synthesized in solution, indicated the authenticity and suitability of the designed ligand for the purification of immunoglobulins. The immobilized ligand displayed an apparent static binding capacity of ~17 mg IgG ml(-1) and a dissociation constant of 5.34 × 10(-5)  M. Preparative chromatography demonstrated the ability of the immobilized ligand to purify IgG and Fab fragments from crude mammalian and yeast cell cultures, under near physiological ionic strength and pH, to yield proteins of 99% and 93% purity, respectively.
authors: Institute of Biotechnology, Department of Chemical Engineering and Biotechnology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QT, UK. ge235@cam.ac.uk
authors: Graziella
authors: G
authors: El Khoury
authors: Christopher R
authors: CR
authors: Lowe
mesh_terms: D000327:Adsorption; D020816:Amino Acid Motifs; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry*; D016466:CHO Cells; D002457:Cell Extracts / Q000302:isolation & purification; D002846:Chromatography, Affinity / Q000379:methods; D006224:Cricetinae; D006863:Hydrogen-Ion Concentration; D055767:Immobilized Proteins / Q000138:chemical synthesis / Q000737:chemistry; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000302:isolation & purification*; D007074:Immunoglobulin G / Q000737:chemistry / Q000302:isolation & purification*; D008024:Ligands; D062105:Molecular Docking Simulation; D018716:Molecular Mimicry; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry*; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D012685:Sepharose / Q000737:chemistry; D060327:Solid-Phase Synthesis Techniques; D015003:Yeasts
chemical_list: D001426:Bacterial Proteins; D002457:Cell Extracts; C034284:IgG Fc-binding protein, Streptococcus; D055767:Immobilized Proteins; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D008024:Ligands; D010446:Peptide Fragments; D012685:Sepharose