Efficient selection of IgG Fc domain-binding peptides fused to fluorescent protein using E. coli expression system and dot-blotting assay.
Antibody purification technology is of particular industrial importance due to the rapidly increasing use of antibodies in protein purification, diagnostic and therapeutic applications. Such purification has mostly relied on affinity chromatography using Protein A or Protein G as affinity ligands. Several synthetic ligands have also been developed to overcome the disadvantages associated with protein affinity ligands, which include high cost, low stability and possible contamination if the proteins have been expressed in bacteria. In the present study, a convenient selection method for new peptides binding to the IgG Fc domain was developed. The method includes the construction of a DNA library fused to the 5'-position of the eGFP gene expressed from a constitutive vector, expression of the library in Escherichia coli, fluorescence-based screening, and determination of the antibody-binding affinities of selected peptides using surface plasmon resonance. With this method, five novel peptides were identified as new affinity ligands for the IgG Fc domain, and the binding affinities were appropriate for antibody purification. This method is a convenient alternative to phage or bacterial surface display and can be used in the routine biochemistry laboratory.
Jeong
Yu-jin
YJ
BioNanotechnology Research Center, KRIBB and Nanobiotechnology Division of UST Daejeon, Republic of Korea.
Kang
Hyo Jin
HJ
Bae
Kwang-Hee
KH
Kim
Min-Gon
MG
Chung
Sang J
SJ
D000595:Amino Acid Sequence; D000818:Animals; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D006041:Goats; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting / Q000379:methods*; D007141:Immunoglobulin Fc Fragments / Q000378:metabolism*; D007074:Immunoglobulin G / Q000378:metabolism*; D051379:Mice; D009842:Oligopeptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019151:Peptide Library; D011485:Protein Binding / Q000276:immunology; D011817:Rabbits; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D020349:Surface Plasmon Resonance
D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D009842:Oligopeptides; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins