Yeast surface display for directed evolution of protein expression, affinity, and stability.
The described protocols enable thorough screening of polypeptide libraries with high confidence in the isolation of improved clones. It should be emphasized that the protocols have been fashioned for thoroughness, rather than speed. With library plasmid DNA in hand, the time to plated candidate yeast display mutants is typically 2-3 weeks. Each of the experimental approaches required for this method is fairly standard: yeast culture, immunofluorescent labeling, flow cytometry. Protocols that are more rapid could conceivably be developed by using solid substrate separations with magnetic beads, for instance. However, loss of the two-color normalization possible with flow cytometry would remove the quantitative advantage of the method. Yeast display complements existing polypeptide library methods and opens the possibility of examining extracellular eukaryotic proteins, an important class of proteins not generally amenable to yeast two-hybrid or phage display methodologies.
Boder
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Department of Chemical Engineering, University of Pennsylvania, Philadelphia 19104, USA.
Wittrup
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D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D015951:Factor Xa / Q000737:chemistry / Q000235:genetics*; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics*; D000072235:Mating Factor; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016366:Open Reading Frames; D019151:Peptide Library; D010455:Peptides / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D017931:DNA Primers; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D010455:Peptides; D011993:Recombinant Fusion Proteins; D000072235:Mating Factor; D015951:Factor Xa