A biomimetic Protein G affinity adsorbent: an Ugi ligand for immunoglobulins and Fab fragments based on the third IgG-binding domain of Protein G

publication · 7 years ago
by Graziella El Khoury, Christopher R. Lowe (University of Cambridge)
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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 chromatograph y 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.

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