Post Doctoral Fellow

Cell Biology

Harvard Medical School (Boston, MA)

Spyros Artavanis-Tsakonas

Elucidation of the Protein Interactome of Drosophila melanogaster

Key functional aspects of the cellular machinery are driven by groups of proteins assembled into macromolecular complexes, or “molecular machines.” In order to generate a protein complex map of the Drosophila melanogaster protein-protein interaction network, we have undertaken affinity-purification of protein complexes using >5,000 bait proteins expressed in fusion with a FLAG-HA moiety. The major steps in this process are: I) systematic molecular cloning of protein-encoding ORFs into our FLAG-HA expression vector; II) recombinant expression of the bait proteins in cell culture; III) co-affinity purification of each bait protein with its interacting partners; IV) mass spectrometric analysis of the protein complexes to identify constituent members; and V) extensive bioinformatics analysis and comparison with prior knowledge. Multi-level analysis of the data will reveal how the ensemble of cellular proteins partitions into functional complexes. We have purified several well-known complexes and identified many new members. Our effort also has the potential to identify previously uncharacterized protein complexes and to define the sub-cellular context or new molecular functions for a majority of the proteins. These results will be accessible through Flybase. Data integration with other functional attributes will allow greater and more complete insight into the cellular machinery. As an additional resource for the community, we are generating stable transgenic lines for the same set of tagged proteins where expression is driven by a UAS promoter, to allow in vivo expression of the protein in a spatio-temporally regulated manner for further analysis.

2005-2007 Project Associate, NCBS, INDIA