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In order to characterize in vivo interactions in Drosophila melanogaster, and further our understanding of developmental processes, hybrid PiggyBac/P-element YFP traps with Strep Tag affinity tags were generated to isolate multi-protein complexes. The incorporated Strep affinity tag allows the target protein to be isolated from its native environment, by tandem affinity purification, along with any associating proteins. Purification efficiency can be followed visually by the YFP marker. Complex proteins are then identified by tandem mass-spectrometry against the Drosophila melanogaster database using the Mascot search engine.
Due to the vast number of putative proteins, a high throughput purification and identification system has to be adopted to maximize coverage. Protein extraction techniques have been optimized to include isolation of tagged membrane proteins whilst maintaining their native structure and complexes. Strep-Tactin columns have proven to be inefficient at eluting high enough yields of Strep-tagged purified protein thus making the purification time consuming and tedious. However, modifying elution protocols has enabled us to load eluates directly onto the Mass Spec and thus identify all proteins in a single run.
The addition of a FLAG tag will also greatly improve the purification efficiency. Vectors have been designed and new fly lines are underway.
Mass Spec results were confirmed by visualizing the corresponding sized proteins on SDS-PAGE gels and western blotting using a GFP antibody to identify the tagged bait. Data from this analysis will be uploaded onto a worldwide accessible database and supersede data generated from the yeast two-hybrid interaction screens.