Cyclic peptide ligand with high binding capacity for affinity purification of immunoglobulin G.
The rapidly increasing implementation of antibodies in therapeutic and diagnostic applications has necessitated the development of antibody production and purification technologies for both academic and industrial usage. Bacterial Protein A and Protein G are known to bind antibodies with high affinity and have facilitated the isolation and purification thereof. Recently, small peptide ligands (i.e. IgG Fc domain-binding peptides, FcBP) that specifically bind to the Fc-domain of antibodies were reported. In the present study we describe the development of a reusable high affinity column for antibody purification utilizing immobilized FcBP, comprising 13 amino acids residues, on a sepharose resin. In addition to FcBP, Cys to Ser substituted FcBP (FcBP-Ser), reduced FcBP (FcBP-Red), commercial Protein A and Protein G resins, packed into columns, were evaluated for antibody purification. All these columns except the FcBP-Ser one showed good binding capacity for a humanized IgG (trastuzumab) and a chimeric IgG (cetuximab). The column packed with FcBP-Red allowed antibody purification at a less acidic pH (pH 4.8) than was required for the other ligand affinity columns used in our experiments (i.e., pH 3.2 for Protein G and FcBP columns, and pH 3.5 for Protein A column, respectively). Utilizing the FcBP column, antibodies from swine human sera were isolated with a purity of 95%. Interestingly, the FcBP column could be easily regenerated and operated without loss of efficiency for up to 60 runs, the maximum number of runs performed in the present study.
Research Institute of Biomolecular Chemistry, Dongguk University, Seoul 100-715, Republic of Korea.
Hyo Jin
HJ
Kang
Department of Chemistry, Seoul National University, Seoul 151-742, Republic of Korea.
Weonu
W
Choe
Biotherapeutics Translational Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-806, Republic of Korea.
Jeong-Ki
JK
Min
BioNanotechnology Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-806, Republic of Korea.
Young-Mi
YM
Lee
Department of Chemistry, Seoul National University, Seoul 151-742, Republic of Korea. Electronic address: kimbm@snu.ac.kr.
B Moon
BM
Kim
Research Institute of Biomolecular Chemistry, Dongguk University, Seoul 100-715, Republic of Korea; Department of Chemistry, Dongguk University, Seoul 100-715, Republic of Korea. Electronic address: sjchung@dongguk.edu.
Sang J
SJ
Chung
D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry; D002846:Chromatography, Affinity; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000302:isolation & purification* / Q000378:metabolism; D008024:Ligands; D010456:Peptides, Cyclic / Q000378:metabolism*; D011485:Protein Binding; D012685:Sepharose / Q000737:chemistry; D013205:Staphylococcal Protein A / Q000737:chemistry; D013552:Swine
D001426:Bacterial Proteins; D007074:Immunoglobulin G; D008024:Ligands; D010456:Peptides, Cyclic; D013205:Staphylococcal Protein A; D012685:Sepharose
Affinity column; Antibody purification; IgG Fc domain-binding peptide (FcBP); Protein A; Protein G