A New Ligand for Immunoglobulin G Subdomains by Screening of a Synthetic Peptide Library

Abstract

By screening a synthetic peptide library of general formula (NH(2)-Cys1-X2-X3-X4)(2)-Lys-Gly-OH, a disulfide-bridged cyclic peptide, where X2-X3-X4 is the tripeptide Phe-His-His, has been selected as a ligand for immunoglobulin G (IgG). The peptide, after a preliminary chromatographic characterization, has proved useful as a new affinity ligand for the purification of polyclonal as well as monoclonal antibodies from biological fluids, with recovery yields of up to 90% (90% purity). The ligand is able to bind antibody fragments containing both Fab and Fc from different antibody isotypes, a fact suggesting the presence of at least two different antibody-binding sites. While the recognition site on Fab is unknown, comparative binding studies with Fc, in association with the striking similarities of the peptide (named Fc-receptor mimetic, FcRM) with a region of the human FcgammaRIII receptor, strongly indicate that the peptide could recognize a short amino acid stretch of the lower hinge region, which has a key role in autoimmune disease triggering. The unique properties make the ligand attractive for both the purification of antibody fragments and as a lead for the generation of Fc-receptor antagonists.

Fc-Binding Ligands of Immunoglobulin G: An Overview of High Affinity Proteins and Peptides

“…Immobilized APAR on agarose showed a maximum Ab binding capacity of 9.1 mg/mL and gave electrophoretically pure anti-Granulocyte macrophage-colony stimulating factor monoclonal Ab (produced in mouse) in a yield of 95%. Verdoliva et al described the disulfide bridged synthetic peptide (NH 2 -Cys-Phe-His-His) 2 -Lys-Gly-OH, denoted as FcRM, as a ligand for IgG [54]. The dissociation constant of FcRM was reported to be 20 μM and FcRM proved useful as an affinity ligand in the purification of IgG.…”

“…The reported affinity constants are towards IgG. Elution pH of antibody from affinity ligand column: PAM, 3 or 9; Fc-III, 3.5; Fc-III-4C, 3.5; FcRM, 2.7 [42,45,50,51,54]. …”

Discovery and Characterization of a Peptide Motif That Specifically Recognizes a Non-native Conformation of Human IgG Induced by Acidic pH Conditions

“…Ehrlish and Bailon, and Krook et al also reported IgGbinding peptides discovered using the filamentous phage display technique (5,6). Recently, Verdoliva et al screened a synthetic peptide library and identified an IgG-binding cyclic dimeric peptide that recognized the lower hinge region of IgG (7,8). Of these earlier attempts, the Fc-III peptide with an intramolecular disulfide bond reported by DeLano et al in 2000 (9) is a potential candidate that can displace Protein A functions.…”

Binding site on human immunoglobulin G for the affinity ligand HWRGWV

“…The cyclic Fc-III peptide (DCAWHLGELVWCT) (DeLano et al ., 2000), identified by screening a phage display library for binding to Fc, was found to compete effectively with Protein A. Crystallography studies showed that the peptide targeted the C H 2-C H 3 interface of Fc. The cyclic tripeptide (CFHH) 2 KG, selected against mouse IgG and named FcRM, resembled the loop structure of Fc-RIII in binding the lower hinge region (Pro230–Ser239) of the Fc fragment as determined by nuclear magnetic resonance (NMR) (Verdoliva et al ., 2005). Linear peptides with sequences taken from the loop residues of the Fc receptor for bovine IgG2 were tested and modified, with the minimum sequence Phe82–Val85 (FIGV) finally identified for its ability to inhibit the binding of the receptor to bovine IgG2 (Zhang et al ., 2006).…”