Development of the Double Cyclic Peptide Ligand for Antibody Purification and Protein Detection

Abstract

Development of a peptide-based affinity matrix and detection reagent is important for biomedical research and the biopharmaceutical industry. In the present work, we designed and synthesized an immunoglobin G (IgG)-binding peptide ligand, Fc-III-4C. Fc-III-4C is composed of 15 residues, and the 4 cysteine residues form 2 disulfide bonds to generate a double cyclic structure. The binding affinity of the Fc-III-4C peptide toward human IgG was determined to be 2.45 nM (Kd), which is higher than that of IgG with Protein A/G (Pro-A/G). Importantly, the Fc-III-4C peptide displayed high affinity to various IgGs from different species. Fc-III-4C immobilized agarose beads exhibited high stability and reusability when compared with that of the Pro-A/G-immobilized beads. The conjugate of Fc-III-4C with FITC was demonstrated to be suitable for immunofluorescence detection of proteins expressed in cells. These results demonstrate that the Fc-III-4C peptide is a useful affinity ligand for antibody purification and as a protein detection reagent.

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

“…Gong et al reported a bicyclic derivative of Fc-III, based on the findings of Dias et al (described above) detailing the enhanced binding affinity of the double cyclic FcBP-2 [51]. It was proposed that the incorporation of the d -Pro- l -Pro in addition to the N to C termini cyclization to construct FcBP-2 would present various difficulties in the recombinant production thereof.…”

“…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]. …”

Peptide‐Directed DNA‐Templated Protein Labelling for The Assembly of a Pseudo‐IgM

“…FC‐III has a low affinity (greater than 20 μ m ) towards non‐human IgGs, making it unsuitable for DNA‐templated reactions with these targets . An expanded bicyclic version of the peptide, the FC‐III 4c peptide, has a greater affinity towards various non‐human IgGs and proved suitable to guide the DNA‐templated reaction towards murine anti‐β‐tubulin IgG1 when employed at a 1:1 ratio as analyzed by SDS‐PAGE (Supporting Information, Figure S16). The purified products of all DNA–protein conjugates were obtained by anion exchange HPLC purification and the identity was confirmed by MALDI‐TOF MS (Supporting Information, Figure S18–21).…”

Fc-binding antibody-recruiting molecules exploit endogenous antibodies for anti-tumor immune responses

“…29 However, recruited antibodies did not mediate ADCC under the previous experimental conditions. Given that ADCC is regulated by the overall affinity of the antibody to its antigen 33 and CD16a, 34 we hypothesized that the replacement of the Fc-III peptide with a recently reported peptide, Fc-III4C, which has higher affinity for IgG-Fc, 35 would strengthen the recruited antibody ability to activate effector cells such as NK cells for target cell destruction. We conducted quantitative evaluations of antibody recruitment and determined the anti-tumor effects of Fc-ARM both in vitro and in vivo to prove the concept of the Fc-ARM strategy.…”