Yeast TwoHybrid system

1
Improved!

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

Maarten de Smit m.smit_at_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
gt 5.000.000 transformants needed
bait plasmid
5.000.000 prey-transformants purchased
mating
gt 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 1104 cfu/µg Empty yeast
prey vector 2105 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
Wanted controllable promoter for bait
bait plasmid
prey library
off
off
Bait promoter off yeast happy efficient
transformation
Bait promoter on bait expressed two-hybrid
selection
on
8
Copper-controlled promoters

• Copper inductionCUP1-CYC1fusion promoter
• Copper repressionCTR3-CYC1fusion promoter

reporter fusion GAL4BD - p53
9
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
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
on
off
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