Developing novel state-of-the-art protein-protein interaction screens in mammalian cells
Friday 23 January 2009
Prof.dr. Paul Coffer and dr. Jeffrey Beekman
Project
Cellular functions are organized through complex arrays of protein-protein interactions. Over the past 15 years, multiple screens have been developed to identify novel protein-protein interactions. Most of these screens, including the successful yeast two-hybrid system, are protein fragment complementation assays (PCA). In PCA, protein interactions are reported by protein activity (a transcription factor in case of the yeast two-hybrid system) which is only present when a pair of proteins interacts. This is accomplished by dissecting a reporter protein into two non-functional, non-interacting parts and fusing these parts to possibly interacting proteins. If expression of these fusion proteins results in reporter activity, the proteins are believed to interact and verification procedures can be initiated. In this way, particular proteins (baits) can be screened against cDNA libraries. Although the yeast-two-hybrid system is commonly used, this system has severe limitations: proteins are overexpressed in yeast cells, interacting protein pairs need to translocate to the nucleus to activate transcription, and proteins with transcriptional activity frequently result in false-positive hits. Novel systems have now been developed that allows the screening of cDNA libraries in mammalian cells using fluorescent proteins (such as YFP) as reporters, however, these screens still depend on the quality of the cDNA library and overexpression of the protein pairs. In this student project, a novel state-of-the-art retroviral PCA system will be tested using syntenin and SOX-4 as bait proteins and an enhanced YFP version as reporter. First, cell clones need to be generated that expresses low levels of the bait proteins fused to the first part of YFP (YFP1). Subsequently, these cells are infected with retroviral cassettes containing an inducible promoter, the second YFP fragment (YFP2), and a 3’ splice donor site. Upon induction of promoter activity, mRNA transcripts are then produced in which YFP2 acts as a first exon that will be spliced in frame with downstream exons resulting in N-terminal YFP2-tagging of cellular proteins. Using high throughput cell sorting (~20.000 cells per second), highly fluorescent cells can be sorted in single wells allowing their expansion and characterization of the retroviral integration site to identify the protein that interacts with the bait. In these clones, the subcellular localization of the interaction can be analyzed, the interaction can be verified by co-immunoprecipitation, and the capacity of relevant stimuli (such as IL-5) to modulate the interaction can be assessed. This system would facilitate quick identification and verification of novel interacting protein pairs.
Research questions:
Can retroviral PCA identify known and novel interacting proteins of syntenin and SOX-4?
What controls can be designed to minimize false-positive hits?
Can positive hits be verified using a panel of stimuli and pharmacological inhibitors?
Techniques
A variety of molecular and cell biological techniques including cloning, transfection, viral transduction, cell sorting
Duration
6 or 9 months
Contact
Dr. Jeffrey Beekman, j.beekman@umcutrecht.nl
Prof.dr. Paul Coffer, p.j.coffer@umcutrecht.nl
Dr. Kristin Denzer, k.denzer@umcutrecht.nl, 088 75 576 73
More info
UMC website - Dept. of Immunology
Molecular immunology website
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