Protocol

cDNA Libraries

Volume 729 of the series Methods in Molecular Biology pp 211-223

Date:

Identification of Protein/Target Molecule Interactions Using Yeast Surface-Displayed cDNA Libraries

  • Scott BidlingmaierAffiliated withUCSF Comprehensive Cancer Center, University of California at San Francisco
  • , Bin LiuAffiliated withDepartment of Anesthesia, UCSF Comprehensive Cancer Center, University of California at San Francisco Email author 

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Abstract

We describe a novel expression cloning method based on screening yeast surface-displayed human cDNA libraries by direct affinity interaction to identify cellular proteins binding to a broad spectrum of target molecules. Being a eukaryote, yeast protein expression pathways are similar to those found in mammalian cells, and therefore, mammalian protein fragments displayed on the yeast cell wall are more likely to be properly folded and functional than proteins displayed using prokaryotic systems. Yeast surface-displayed human cDNA libraries have been successfully used to screen for proteins that bind to posttranslationally modified phosphorylated peptides, small signaling molecule phosphatidylinositides, and monoclonal antibodies. In this article, we describe protocols for using yeast surface-displayed cDNA libraries, coupled with fluorescence-activated cell sorting, to select protein fragments with affinity for various target molecules including posttranslationally modified peptides, small signaling molecules, monoclonal phage antibodies, and monoclonal IgG molecules.

Key words

Yeast surface cDNA display Expression cloning Phage antibody Antigen identification Small molecules Posttranslationally modified ligands