Identification of a ligand for IgG-Fc derived from a soluble peptide library based on fusion proteins secreted by S. cerevisiae.
Biological libraries are important tools in the development of new peptide-based compounds. Here, we describe the use of a soluble peptide library system as a complementary tool in the field of ligand development. Random peptides were expressed in S. cerevisiae as carboxy-terminal extensions of the eukaryotic initiation factor 5a (eIF5a) and secreted into the culture supernatant. Expression and screening of this library were performed in a microwell format. As an example of this versatile approach, we describe the identification of a ligand for the human IgG-Fc fragment. Ligands binding IgG-Fc show great potential in a wide variety of applications including development of therapeutics, streamlining the large-scale purification of antibodies, and applications in diagnostic tests. We demonstrated the utility of this system. After screening only 6160 clones, we identified a ligand with the peptide sequence of TRRRTCSPPTWPRARARSTPSGCSSTGPSANRG. An affinity constant of 3.9 x 10(5) M(-1) was determined by a biosensor method. Handling and maintenance of this library is conceptually simple and highly applicable for automated high-throughput systems.
Mersich
Christa
C
Department of Biotechnology, University of Natural Resources and Applied Life Sciences Vienna, Vienna, Austria.
Billes
Werner
W
Jungbauer
Alois
A
D001681:Biological Assay / Q000379:methods; D015374:Biosensing Techniques / Q000379:methods*; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D007074:Immunoglobulin G / Q000235:genetics / Q000276:immunology*; D008024:Ligands; D019151:Peptide Library; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000276:immunology*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000276:immunology*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000276:immunology*; D012995:Solubility
D007074:Immunoglobulin G; D008024:Ligands; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins