Flow-cytometric isolation of human antibodies from a nonimmune Saccharomyces cerevisiae surface display library
| Autor: | Robert W. Siegel, Yik Andy Yeung, Peter Heinzelman, James R. Coleman, Christilyn P Graff, Lee K. Opresko, H. Steven Wiley, Jennifer R. Cochran, K. Dane Wittrup, David W. Colby, Michael J. Feldhaus, Jeffrey S. Swers, Jane M. Weaver Feldhaus |
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| Rok vydání: | 2003 |
| Předmět: |
Male
Saccharomyces cerevisiae Immunoglobulin Variable Region Biomedical Engineering Bioengineering Yeast display Biology Proteomics Polymerase Chain Reaction Applied Microbiology and Biotechnology Flow cytometry Peptide Library Gene Expression Regulation Fungal medicine Humans Nanotechnology Multiplex Cloning Molecular Peptide library Immunoglobulin Fragments Cells Cultured medicine.diagnostic_test Microchemistry Membrane Proteins Flow Cytometry biology.organism_classification Molecular biology Microspheres Recombinant Proteins Yeast Subcloning Feasibility Studies Molecular Medicine Female Biotechnology |
| Zdroj: | Nature Biotechnology. 21:163-170 |
| ISSN: | 1546-1696 1087-0156 |
| DOI: | 10.1038/nbt785 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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