Abstract
Display on the yeast cell wall is well suited for engineering mammalian cell-surface and secreted proteins (e.g., antibodies, receptors, cytokines) that require endoplasmic reticulum-specific post-translational processing for efficient folding and activity. C-terminal fusion to the Aga2p mating adhesion receptor of Saccharomyces cerevisiae has been used for the selection of scFv antibody fragments with threefold decreased antigen dissociation rate from a randomly mutated library. A eukaryotic host should alleviate expression biases present in bacterially propagated combinatorial libraries. Quantitative flow cytometric analysis enables fine discrimination of kinetic parameters for protein binding to soluble ligands.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, Non-P.H.S.
MeSH terms
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Animals
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Cloning, Molecular
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Endoplasmic Reticulum / metabolism
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Escherichia coli
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Flow Cytometry
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Gene Expression
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Genomic Library
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Immunoglobulin Fragments / biosynthesis*
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Immunoglobulin Fragments / chemistry
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Kinetics
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Mammals
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Mating Factor
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Membrane Fusion
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Mutagenesis
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Peptide Biosynthesis*
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Peptide Library*
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Peptides / chemistry
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Plasmids
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Polymerase Chain Reaction
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RNA Processing, Post-Transcriptional
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Recombinant Fusion Proteins / biosynthesis*
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Recombinant Fusion Proteins / chemistry
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Saccharomyces cerevisiae / genetics*
Substances
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Immunoglobulin Fragments
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Peptide Library
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Peptides
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Recombinant Fusion Proteins
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Mating Factor