Flow-cytometric isolation of human antibodies from a nonimmune Saccharomyces cerevisiae surface display library

• Published in: Nature biotechnology 2003-02, Vol.21 (2), p.163-170
• Main Authors: Feldhaus, Michael J, Wittrup, K. Dane, Siegel, Robert W, Opresko, Lee K, Coleman, James R, Feldhaus, Jane M. Weaver, Yeung, Yik A, Cochran, Jennifer R, Heinzelman, Peter, Colby, David, Swers, Jeffrey, Graff, Christilyn, Wiley, H. Steven
• Format: Article
• Language: English
• Subjects: 60 APPLIED LIFE SCIENCES; AFFINITY; Agriculture; ANTIBODIES; ANTIGENS; b-cells; Bioinformatics; Biological and medical sciences; Biomedical and Life Sciences; Biomedical Engineering/Biotechnology; Biomedicine; Biotechnology; Cells, Cultured; Cloning, Molecular; directed evolution; Feasibility Studies; Female; Flow Cytometry - methods; fragments; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation, Fungal; gene repertoire; Genetic engineering; Genetic technics; Humans; Immunoglobulin Fragments - genetics; Immunoglobulin Fragments - immunology; Immunoglobulin Fragments - isolation & purification; Immunoglobulin Variable Region - genetics; Immunoglobulin Variable Region - immunology; Immunoglobulin Variable Region - isolation & purification; Immunoglobulin Variable Region - metabolism; Life Sciences; Male; Membrane Proteins - genetics; Membrane Proteins - immunology; Membrane Proteins - isolation & purification; Membrane Proteins - metabolism; Methods. Procedures. Technologies; Microchemistry - methods; Microspheres; Miscellaneous; Nanotechnology - methods; Peptide Library; periplasmic expression; phage display, high-affinity; Polymerase Chain Reaction - methods; proteins; PURIFICATION; Reagents; Recombinant Proteins - genetics; Recombinant Proteins - immunology; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; SACCHAROMYCES CEREVISIAE; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - immunology; Saccharomyces cerevisiae - isolation & purification; Saccharomyces cerevisiae - metabolism; selection; SORTING; STABILITY; Synthetic digonucleotides and genes. Sequencing; T-cell receptor; YEASTS
• ISSN: 1087-0156; 1546-1696
• DOI: 10.1038/nbt785

A nonimmune library of 10 9 human antibody scFv fragments has been cloned and expressed on the surface of yeast, and nanomolar-affinity scFvs routinely obtained by magnetic bead screening and flow-cytometric sorting. The yeast library can be amplified 10 10 -fold without measurable loss of clonal diversity, allowing its effectively indefinite expansion. The expression, stability, and antigen-binding properties of >50 isolated scFv clones were assessed directly on the yeast cell surface by immunofluorescent labeling and flow cytometry, obviating separate subcloning, expression, and purification steps and thereby expediting the isolation of novel affinity reagents. The ability to use multiplex library screening demonstrates the usefulness of this approach for high-throughput antibody isolation for proteomics applications.