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Structural Genomics of Pathogenic Protozoa (SGPP) Chris Mehlin, Ph.D. University of Washington

Structural Genomics of Pathogenic Protozoa (SGPP) Chris Mehlin, Ph.D. University of Washington. PI: Wim Hol. University of Washington Seattle Biomedical Research Institute University of Rochester Hauptman-Woodward Medical Research Institute Stanford Synchrotron Radiation Laboratory

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Structural Genomics of Pathogenic Protozoa (SGPP) Chris Mehlin, Ph.D. University of Washington

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  1. Structural Genomics of Pathogenic Protozoa (SGPP) Chris Mehlin, Ph.D. University of Washington PI: Wim Hol University of Washington Seattle Biomedical Research Institute University of Rochester Hauptman-Woodward Medical Research Institute Stanford Synchrotron Radiation Laboratory Advanced Light Source/Berkeley Center for Structural Biology

  2. Trypanosomatids and Plasmodia • Afflict ~500 million people per year • mostly in tropics and subtropics • Cause disability, disfigurement, death • Vaccines not available • Drug resistance is a problem, available drugs toxic • Understudied, especially given human toll

  3. Unusual features of the targeted protozoa • Antigenic variation • Protozoans modify host cells and immune response • Unusual organelles and structures • Rhoptries, Plastids, Glycosomes, Food Vacuoles, Parasitopherous Vacuole, Flagellar Pocket • Trans-splicing of mRNA and lack of introns in trypanosomatids • Unusual RNA processing • Unique transcriptional control, RNA editing

  4. Advantages High likelihood of novel folds No introns in trypanosomatids Challenges 80% A/T content in P. falciparum Introns in P. falciparum difficult to predict Pathogenic Protozoans: Experimental Issues

  5. University of Washington Soluble proteins of trypanosomatids and P. falciparum Protein-protein interactions University of Rochester Soluble proteins of trypanosomatids Membrane proteins Expression Centers

  6. Expression Strategies • Considered two approaches to date • Homologous recombination within E. coli • GTS Inc. system • Too many errors • 2/8 had frameshift mutations • Others had very high rate of mutations within ORF • Range: 2-6 substitutions per 100 bp • Topo cloning

  7. Directional Topo Cloning Proteins will be expressed as a fusion with a minimal N-terminal 6xHis tag: Met-Gly-Ser-Ser-6xHis-Gly-Ser-Pro-Ser-ORF (No protease sites)

  8. Conventional dTopo systems put bulky, hydrophobic A.A. on the N-terminus of the protein due to necessity of Topo binding site (CCCTT) and reading frame chosen. P F T CCC TTC ACC - ORF Directional “flap” We plan to put the directional flap at the other end and change the reading frame of the construct: G S P S GGA TCC CCT TCG-ORF-Stop-GGTG PCR product

  9. Options left open post-cloning: 5’ 3’ G S P S Stop (G) (E) (G) (S) (S) GGA TCC CCT TCG-ORF-TAA GGT GAA GGG ACG TCT GCA C Bam HIPCR productflapSacI Bsg I Bam site should allow us to alter the N-terminal tags. Stop codon can be altered by PCR or cleavage with Bsg I to make a C-terminal fusion.

  10. Thanks to: Mike Gelb Wes Van Voorhis Fred Buckner Wim Hol Min Park Chang Kim Yvonne Rogers Gina McConnell SGPP at University of Washington Terwilliger Lab, Los Alamos Invitrogen

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