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Yeast Two-Hybrid system for working with proteins affecting yeast growth

Improved!. Yeast Two-Hybrid system for working with proteins affecting yeast growth. Maarten de Smit m.smit@chem.leidenuniv.nl. Y2H: Interaction activates reporter. GAL4-DBD- bait (hybrid 1) GAL4-AD- prey (hybrid 2; single or library) bait - prey interaction: GAL4 activates reporter.

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Yeast Two-Hybrid system for working with proteins affecting yeast growth

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  1. Improved! Yeast Two-Hybrid system for working with proteins affecting yeast growth Maarten de Smit m.smit@chem.leidenuniv.nl

  2. Y2H: Interaction activates reporter • GAL4-DBD-bait (hybrid 1) • GAL4-AD-prey(hybrid 2; single or library) • bait-prey interaction: GAL4 activates reporter AD GAL4 DBD DNA GAL1 UAS GAL1 TATA Reporter RNA selectable or detectable protein

  3. Screening a library of prey cDNAs Y2H selective agar plate: activated reporter = growth Colonies contain DNA encoding interacting proteins Sequencing!

  4. Transformation or mating bait plasmid prey library 1 transformant needed > 5.000.000 transformants needed bait plasmid 5.000.000 prey-transformants purchased mating > 5.000.000 diploid clones needed

  5. A bad pilot... Matchmaker™ Two-HybridSystem 3 (Clontech) Bait: SV40 small-t antigenfragment 1-82 ("st1") prey library st1 bait plasmid st1-fusion causes transformation problems? Yeast with bait + prey vector: 1·104 cfu/µg Empty yeast + prey vector: 2·105 cfu/µg

  6. Sick yeast: plasmid loss during growth st1-fusion makes yeast sick: 90% loss of bait plasmid prey library 10% 10% Td = 200 min st1 bait plasmid 90% 90% prey library Td = 100 min

  7. YTH selective plate Wanted: controllable promoter for bait bait plasmid prey library off off Bait promoteroff: yeast happy efficient transformation Bait promoteron: bait expressed two-hybrid selection on

  8. CuSO4 (µM) 0.2 2 20 100 CuSO4 (µM) 0.2 2 20 100 Copper-controlled promoters • Copper induction:CUP1-CYC1fusion promoter • Copper repression:CTR3-CYC1fusion promoter reporter fusion: GAL4BD - p53

  9. 0.7 0.6 CuSO4 (µM) 0.5 0.4 0.2 fraction of cells with plasmid 2 0.3 20 100 0.2 0.1 0 CUP1-CYC1 st1 CTR3-CYC1 st1 ADH1 st1 ADH1 empty bait fusion protein Copper-controlled plasmid loss • Bait fusion-protein down = plasmid loss down plasmid loss measured after overnight growth in selective medium (becomes worse after growth in rich medium before transformation!)

  10. Cu Cu YTH selective plate Copper kills selection... • Copper severely reduces colony formation - from freshly transformed cells - under two-hybrid selective conditions • So we use copper repression, not induction - copper added before library transformation - no copper in selective plates bait plasmid prey library off off on

  11. Preliminary results Preliminary results of pilot screen (small-t 1-82): • screened ~15% of library (human normal keratinocytes) • on ~50 plates (15 cm ) • picked 21 candidates • 13 grow on "medium stringency" selective plates • 2 grow on "high stringency" selective plates Y2H with bait protein affecting yeast growth (plasmid retention) is now feasible

  12. Thanks! Loek Brinkman Patrick Voskamp Mathieu Noteborn

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