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Quy Nguyen and Nora Kabbara 06 March 2014

Quy Nguyen and Nora Kabbara 06 March 2014. Background. How genetic information results in gene products? Human disease exceed number of genes. Disorder based on polygenetic traits. Number of human genes does not differ from other less complex organisms.

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Quy Nguyen and Nora Kabbara 06 March 2014

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  1. QuyNguyen and Nora Kabbara 06 March 2014

  2. Background • How genetic information results in gene products? • Human disease exceed number of genes. • Disorder based on polygenetic traits. • Number of human genes does not differ from other less complex organisms. • C. elegans vs. Humans---> Almost same number of genes yet drastic difference in complexity • Most cellular processes carried out by multiprotein complex. • Use haploid S. cerevisiae (baker’s yeast) to perform comprehensive protein analysis

  3. Methods • Tandem-affinity purification (TAP) • TAP cassette inserted into C terminus of yeast ORF • Generate TAP-tagged fusion protein for purification.

  4. Methods • TAP • TAP Cassette consist of • CBP tag, TEV cleave site, ProtA • Multi-steps purification to yield pure samples under native conditions. Puig, O. et. al. 2001

  5. Methods • Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) • Mass spectrometry for identification of biomolecules that is fragile to conventional ionization. Lewis, K. et. al. 2000

  6. Results

  7. Results

  8. Results • Multiple distinct tags for the protein complex to identify key components. • Identified key subunit (Tpd3) and looked for what other proteins interacted with it. Polyadenylationmachinery complex used as entry point for purification

  9. Results • Understanding integration and coordination of cellular functions. • Different colors show different proteins involved in cellular functions(ex. red for cell cycle, violet energy metabolism).There are even more connections yet to be discovered.

  10. Results

  11. Conclusion • Assign cellular function to new or nonannotated gene products. • Largest analysis of protein complex to date. • TAP can purify complexes from different cellular compartments. • Identification of low-abundance proteins. • Purification of large complex. • Group cellular protein into complexes. • Provide molecular context for choice evaluation of drug targets.

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