A novel mammalian display system for the selection of protein-protein interactions by decoy receptor engagement.
The emerging field of proteomics has created a need for new high-throughput methodologies for the analysis of gene products. An attractive approach is to develop systems that allow for clonal selection of interacting protein pairs from large molecular libraries. In this study, we have characterized a novel approach for identification and selection of protein-protein interactions, denoted SPIRE (selection of protein interactions by receptor engagement), which is based on a mammalian expression system. We have demonstrated proof of concept by creating a general plasma membrane bound decoy receptor, by displaying a protein or a peptide genetically fused to a trunctated version of the CD40 molecule. When this decoy receptor is engaged by a ligand to the displayed protein/peptide, the receptor expressing cell is rescued from apoptosis. To design a high-throughput system with a highly parallel capacity, we utilized the B cell line WEHI-231, as carrier of the decoy receptor. One specific peptide-displaying cell could be identified and amplified, based on a specific receptor engagement, in a background of 12 500 wild-type cells after four selections. This demonstrates that the approach may serve as a tool in post-genomic research for identifying protein-protein interactions, without prior knowledge of either component.
Ellmark
Peter
P
Department of Immunotechnology, Lund University, Lund, Sweden.
Ohlin
Mats
M
Borrebaeck
Carl A K
CA
Furebring
Christina
C
D000818:Animals; D017209:Apoptosis; D019013:CD40 Antigens / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D049109:Cell Proliferation; D003001:Cloning, Molecular / Q000379:methods*; D015870:Gene Expression; D006801:Humans; D008024:Ligands; D008322:Mammals / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D019151:Peptide Library; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
D019013:CD40 Antigens; D008024:Ligands; D019151:Peptide Library; D011993:Recombinant Fusion Proteins