Human IgA-binding peptides selected from random peptide libraries: affinity maturation and application in IgA purification.
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.
Graduate School of Science and Engineering, Kagoshima University, Kagoshima 890-0065, Japan.
Takaaki
T
Hatanaka
Shinji
S
Ohzono
Mirae
M
Park
Kotaro
K
Sakamoto
Shogo
S
Tsukamoto
Ryohei
R
Sugita
Hiroyuki
H
Ishitobi
Toshiyuki
T
Mori
Osamu
O
Ito
Koichi
K
Sorajo
Kazuhisa
K
Sugimura
Sihyun
S
Ham
Yuji
Y
Ito
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000915:Antibody Affinity / Q000276:immunology*; D002846:Chromatography, Affinity; D017124:Conserved Sequence; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007070:Immunoglobulin A / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011961:Receptors, Fc / Q000737:chemistry; D015203:Reproducibility of Results; D017386:Sequence Homology, Amino Acid; D013816:Thermodynamics
D007070:Immunoglobulin A; D019151:Peptide Library; D010455:Peptides; D011961:Receptors, Fc