title: In vitro affinity screening of protein and peptide binders by megavalent bead surface display.
abstract: The advent of protein display systems has provided access to tailor-made protein binders by directed evolution. We introduce a new in vitro display system, bead surface display (BeSD), in which a gene is mounted on a bead via strong non-covalent (streptavidin/biotin) interactions and the corresponding protein is displayed via a covalent thioether bond on the DNA. In contrast to previous monovalent or low-copy bead display systems, multiple copies of the DNA and the protein or peptide of interest are displayed in defined quantities (up to 10(6) of each), so that flow cytometry can be used to obtain a measure of binding affinity. The utility of the BeSD in directed evolution is validated by library selections of randomized peptide sequences for binding to the anti-hemagglutinin (HA) antibody that proceed with enrichments in excess of 10(3) and lead to the isolation of high-affinity HA-tags within one round of flow cytometric screening. On-bead K(d) measurements suggest that the selected tags have affinities in the low nanomolar range. In contrast to other display systems (such as ribosome, mRNA and phage display) that are limited to affinity panning selections, BeSD possesses the ability to screen and rank binders by their affinity in vitro, a feature that hitherto has been exclusive to in vivo multivalent cell display systems (such as yeast display).
authors: Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, CB2 1GA Cambridge, UK.
authors: Letizia
authors: L
authors: Diamante
authors: Pietro
authors: P
authors: Gatti-Lafranconi
authors: Yolanda
authors: Y
authors: Schaerli
authors: Florian
authors: F
authors: Hollfelder
mesh_terms: D000595:Amino Acid Sequence; D061505:Cell Surface Display Techniques / Q000379:methods*; D004247:DNA / Q000235:genetics; D005838:Genotype; D008863:Microspheres; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010641:Phenotype; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
chemical_list: D010455:Peptides; D011506:Proteins; D004247:DNA
keywords: antibody; directed evolution; emulsion PCR; phage display; protein display
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