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Expanding the Pool

Expanding the Pool. Characterizing LAGLIDADG Homing Endonuclease Orthologs. Picking the ORF Regions to Use. OK active sites 50-60+ % homology Regions homologous to Ani Optimization Yeast expression, restriction Surface expression?. Intron. ORF. Expression & Relatedness.

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Expanding the Pool

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  1. Expanding the Pool Characterizing LAGLIDADG Homing EndonucleaseOrthologs

  2. Picking the ORF Regions to Use • OK active sites • 50-60+ % homology • Regions homologous to Ani • Optimization • Yeast expression, restriction • Surface expression? Intron ORF

  3. Expression & Relatedness

  4. Expression problems • Mutagenesis & directed evolution to regain & improve expression • Glycosylation changes? • Tas Tin and Vin are predicted to be lacking glycosylation in regions glycosylated in Pno, Ach, Hje, and Ani • Tas and Tin are predicted to be glycosylated at DNA binding regions • Poor surface expressers but otherwise OK

  5. Target Determination • Target for Ani at intron/exon junctions • By Homology… • Central 4 are hard to predict TGAGGAGGTT T CT CTGTAAA TGGGGAGG TTT TTCAGTATC

  6. Binding Vs Ani Target &Predicted Targets Conclusion: Tas, Tin, and Vin are not producing a viable surfac-expressed HE

  7. Cleavage Ach Hje Pno Ani E148D Ach Hje Pno Ach Hje Pno Ani 37°C, against Predicted Targ 37°C, against AniTarg 30°C, against Predicted Targ Target Only

  8. What Changes Are Responsible? • Variance at DNA-interacting domains • Insertions relative to Ani

  9. Differences at DNA-Binding Regions • 5Å interactions as spheres • Unchanged residues in green • Conserved Residues in Purple +5, +9 & +10 positions (white) +2 & +5 positions (white)

  10. Insertions • Directly change DNA-interacting regions (left) • May realign directly interacting regions (right) • Harder to obtain by directed evolution alone +5, +9 & +10 positions (white) -8 position (white)

  11. Rosetta Predictions • -8 A -> G • K24N & T29K • D73N Conserved residue changed

  12. Take-home Message Future Directions • We can use homology searches to find functional homing endonucleaseswith different targets • This can help us determine what AA changes affect what target specificities • Mutagenesis & directed evolution to improve surface expression and/or activity • Comparing Ani ability to cleave predicted targets to the corresponding enzyme’s cleavage to evaluate actual changes • Determine specificity with 1-off panels • DNA shuffling to generate hybrid HE’s • Begin to look at which changes are tolerated and which aren’t

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