Human IgA-binding Peptides Selected from Random Peptide Libraries: AFFINITY MATURATION AND APPLICATION IN IGA PURIFICATION

Takaaki Hatanaka,Shinji Ohzono,Mirae Park,Kotaro Sakamoto,Shogo Tsukamoto,Ryohei Sugita,Hiroyuki Ishitobi,Toshiyuki Mori,Osamu Ito,Koichi Sorajo,Kazuhisa Sugimura,Sihyun Ham,Yuji Ito

doi:10.1074/jbc.m112.389742

Abstract

Phage display system is a powerful tool to design specific ligands for target molecules. Here, we used disulfide-constrained random peptide libraries constructed with the T7 phage display system to isolate peptides specific to human IgA. The binding clones (A1-A4) isolated by biopanning exhibited clear specificity to human IgA, but the synthetic peptide derived from the A2 clone exhibited a low specificity/affinity (K(d) = 1.3 μm). Therefore, we tried to improve the peptide using a partial randomized phage display library and mutational studies on the synthetic peptides. The designed Opt-1 peptide exhibited a 39-fold higher affinity (K(d) = 33 nm) than the A2 peptide. An Opt-1 peptide-conjugated column was used to purify IgA from human plasma. However, the recovered IgA fraction was contaminated with other proteins, indicating nonspecific binding. To design a peptide with increased binding specificity, we examined the structural features of Opt-1 and the Opt-1-IgA complex using all-atom molecular dynamics simulations with explicit water. The simulation results revealed that the Opt-1 peptide displayed partial helicity in the N-terminal region and possessed a hydrophobic cluster that played a significant role in tight binding with IgA-Fc. However, these hydrophobic residues of Opt-1 may contribute to nonspecific binding with other proteins. To increase binding specificity, we introduced several mutations in the hydrophobic residues of Opt-1. The resultant Opt-3 peptide exhibited high specificity and high binding affinity for IgA, leading to successful isolation of IgA without contamination.

Highlights

Pharmaceutical application of human IgA requires a highly specific IgA purification system
To design a peptide with increased binding specificity, we examined the structural features of Opt-1 and the Opt-1-IgA complex using all-atom molecular dynamics simulations with explicit water
Five rounds of biopanning were performed against human IgA (hIgA) using T7 phage-displayed random peptide libraries of X3CX7–10CX3, where X represents randomized amino acid positions

Summary

Background

Pharmaceutical application of human IgA requires a highly specific IgA purification system. Conclusion: The designed IgA-binding peptide has high affinity and specificity for human IgA. The M proteins from S. pyogenes, M22, Arp, or Sir, exhibit binding activities toward IgA1 and A2 or the monomeric and secretory forms of IgA. The use of bacterial proteins for pharmaceutical antibody purification requires careful attention to prevent contamination with endotoxin or bacterial proteins, given their highly toxic and antigenic nature, as described for the protein A/G column used in IgG purification for pharmaceutical use [23] To solve this problem, synthetic low molecular weight ligands, such as TG19318 [24] and protein A mimetic compounds [25], have been investigated. Sandin et al [26] reported the purification of hIgA using a synthetic peptide comprising 50 residues extracted from the IgA-binding domain (Sap) of the Sir M protein. Our peptide is only 16 residues long and exhibits high specificity/ affinity for hIgA, which is suitable for hIgA purification

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION