Yeast Two-Hybrid system

for working with proteins

affecting yeast growth

Maarten de Smitm.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

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!