yeast two-hybrid system for working with proteins affecting yeast growth maarten de smit...
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Yeast Two-Hybrid system
for working with proteins
affecting yeast growth
Maarten de [email protected]
Y2H: Interaction activates reporter
• GAL4-DBD-bait (hybrid 1)
• GAL4-AD-prey (hybrid 2; single or library)
• bait-prey interaction: GAL4 activates reporter
GAL1 UAS GAL1 TATA Reporter
GAL4
DNA
RNA
selectableor detectableprotein
DBD
AD
Screening a library of prey cDNAs
Y2H selective agar plate: activated reporter = growth
Colonies contain DNA encoding interacting proteinsSequencing!
Transformation or mating
baitplasmid
preylibrary
1 transformantneeded
> 5.000.000 transformants needed
baitplasmid
5.000.000prey-transformants
purchased
mating
> 5.000.000 diploidclones needed
A bad pilot...
st1 baitplasmid
preylibrary
Matchmaker™ Two-HybridSystem 3 (Clontech)
Bait: SV40 small-t antigenfragment 1-82 ("st1")
st1-fusion causes transformation problems?Yeast with bait + prey vector: 1·104 cfu/µgEmpty yeast + prey vector: 2·105 cfu/µg
Sick yeast: plasmid loss during growth
preylibrary
preylibrary
10%
90%
10%
90%
st1-fusion makes yeast sick:90% loss of bait plasmid
st1 baitplasmid
Td = 200 min
Td = 100 min
Wanted: controllable promoter for bait
baitplasmid
preylibrary
Bait promoter off: yeast happyefficient transformation
Bait promoter on: bait expressedtwo-hybrid selection
YTH selective plate
off off
on
Copper-controlled promoters
• Copper induction:CUP1-CYC1fusion promoter
• Copper repression:CTR3-CYC1fusion promoter
0.2 2 20 100
CuSO4 (µM)
0.2 2 20 100
CuSO4 (µM)
reporter fusion: GAL4BD - p53
Copper-controlled plasmid loss• Bait fusion-protein down = plasmid loss down
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
CUP1-CYC1st1
CTR3-CYC1st1
ADH1st1
ADH1empty
0.2
2
20
100
CuSO4
(µM)
fra
ctio
n o
f ce
lls w
ith p
lasm
id
bait fusionprotein
plasmid loss measured after overnight growth in selective medium(becomes worse after growth in rich medium before transformation!)
• 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
Copper kills selection...
Cu
baitplasmid
preylibrary
YTH selective plate
Cu
off onoff
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
Preliminary results
Thanks!