The powerful combination of phage surface display of cDNA libraries and high throughput screening.
Phage surface display of cDNA libraries facilitates cloning, expression and rapid selection of functional gene products physically linked to their genetic information through gene product-ligand interactions. Efficient screening technologies based on selective enrichment of clones expressing desired gene products allows, within a short time, the isolation of all ligand-specific clones that are present in a library. Manual identification of clones by restriction analysis and random sequencing is unlike to be successful for the isolation of gene products derived from rare mRNA species resulting from selection of the libraries using polyvalent ligands like serum from patients. Here we describe rapid handling of large numbers of individual clones selected from molecular libraries displayed on phage surface using the power of robotics-based high throughput screening. The potential of the combination of cDNA-phage surface display, with selection for specific interactions by functional screening and robotic technology is illustrated by the isolation of more sequences potentially encoding IgE-binding proteins than postulated from Western blot analyses using extracts derived from raw material of complex allergenic sources. The subsequent application of functional enrichment and robotics-based screening will facilitate the rapid generation of information about the repertoire of protein structures involved in allergic diseases.
Crameri
R
R
Swiss Institute of Allergy and Asthma Research (SIAF, Obere Strasse 22, Davos, CH-7270, Switzerland. crameri@siaf.unizh.ch
Kodzius
R
R
D001435:Bacteriophages / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods*; D020650:Combinatorial Chemistry Techniques; D018076:DNA, Complementary
D018076:DNA, Complementary