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Single clone expression cloning and protein microarray platforms for novel extracellular receptor‐ligand identification
Author(s) -
Tom Irene,
Quis Gabriel,
Estevez Alberto,
Paluch Maciej,
Bowman Krista,
Franke Yvonne,
Billeci Karen,
Hass Phil,
Tang Jerry,
LewinKoh Nicholas,
Eaton Dan,
Stephan JeanPhilippe,
Gonzalez Lino
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.803.5
Subject(s) - fusion protein , clone (java method) , cloning (programming) , protein microarray , biology , expression cloning , microarray , computational biology , transmembrane protein , protein–protein interaction , microbiology and biotechnology , gene , genetics , gene expression , receptor , complementary dna , recombinant dna , computer science , programming language
Here we describe the development of two complementary high‐throughput screening technologies consisting of single clone expression cloning and protein microarray platforms that permit genome‐wide screening of a protein of interest against thousands of interactions either displayed on a cell surface or on a microarray. The single clone expression cloning platform is fully automated and capable of efficient transfection, expression, and binding analysis of a bait protein to cells transfected with individual clones from the clone library. For low affinity interactions, we generated bait protein with an Fc fusion to increase the avidity. Our secreted protein microarray has over 1500 secreted or extracellular domain of single transmembrane human proteins represented to enable simultaneous systematic probing of a bait protein against proteins on the microarray. To enhance detection of low affinity interactions, we assembled bait Fc fusion proteins into multivalent complexes with protein A microbeads. As proof of concept, we evaluated well‐characterized extracellular receptor‐ligand interactions with diverse binding affinities on the single clone expression cloning and protein microarray platforms. Overall, we found that the interactions identified using both of our screening methodologies were highly specific and displayed minimal off‐target binding. Furthermore, we demonstrate the utility of both technologies for novel receptor‐ligand discovery.