Yeast-two hybrid

Yeast 2 Hybrid

The objective of the yeast 2-hybrid (Y2H) technique is to screen for or to examine protein-protein interactions. Briefly, a transcription factor (TF) is separated into its DNA binding domain (DBD) and RNA-polymerase activating domain (AD) and separated like this the TF is not active. Now the protein to study is cloned in frame with the DBD (also called "bait") and the second protein (or a cDNA library in the case of a screening) is cloned in frame with the AD (also called "prey"). Each of these chimeric proteins do not act as a functional TF - but when there are protein-protein interactions, then the two parts of the TF are coming closed together and act again as a TF - in this case for reversing auxotrophy, e.g. His, and for a reporter gene, e.g. β-galactosidase.
A nice overview about the Y2H-system is written by a D. Parchaliuk (Technical Tips Online, 1999):

A nice animation was made by Lodish et al. ©:

Information on yeasts as a model for eucaryonts can be found at Rochester University. The Yeast genome database is curated by Stanford University.

The system used in the CBL is the Hybrid-Hunter from Invitrogen.

The yeast used is Saccharomyces cerevisiae strain L40, which is deposited at ATCC (MYA-3332).
Genotype: MATa his3Δ200 trp1-901 leu2-3112 ade2 LYS::(4lexAop-HIS3) URA3::(8lexAop-LacZ)GAL4
Phenotype: His^-, Trp^-, Leu^-, Ade^-
- Lysine-prototrophy genes fused with 4 x LexA-promotor fused to HIS3: when medium lacks Lys, LYS-genes will be expressed and thus the promotor (LexA) for HIS3 expression is accessible => Strain get His-prototroph when LexA is active (?)
- Uracil3-prototrophy gene fused with 8 x LexA-promotor fused to LacZ: when medium lacks Ura, URA3-gene will be expressed and thus the promotor (LexA) for LacZ expression is accessible => Strain produces β-galactosidase when LexA is active (?)

Bait vector: pHybLex/Zeo (pHL)
- Promotor: truncated ADH1 (alcohol dehydrogenase) for strong constitutive expression of
   - LexA ORF: Complete LexA gene (606 bp)
   - MCS: 9 unique restriction sites: EcoRI, SacI, PvuII, ApaI, Acc65I, NotI, XhoI, SalI, PstI
   - ADH1 transcription termination: stabilises mRNA
- 2µ origin: replication of yeast plasmid (high copy)
- Promotor: TEF1 (transcription elongation factor 1) for expresssion of:
- Promotor: EM7 (synthetic procariontic promotor) for expression of:
   - Sh ble gene (Zeocin resistance)
   - CYC1 transcription termination: stabilises mRNA
- ColE1 origin: replication of bacteria plasmid (E. coli)

Prey vector I: pYESTrp2 (pYT)
- Promotor: GAL1 for strong constitutive expression (within L40) of
   - V5-epitope: for anti-V5 antibodies
   - NLS (nuclear localisation sequence): from SV40 large T antigen
   - B42 ORF
   - MCS: 8 unique restriction sites: HindIII, Acc65I, SacI, BamHI, EcoRI, NotI, XhoI, SphI
   - CYC1 transcription termination: stabilises mRNA
- ColE1 origin: replication of bacteria plasmid (E. coli)
- Ampicillin resistence gene: Selection in E. coli
- TRP1 gene: Auxotrophy selection in trp^- hosts (like L40)
- 2µ origin: replication of yeast plasmid (high copy)
- f1 origin: rescue of ssDNA

Prey vector II: pJG4-5 (pJG)
- Promotor: GAL1 for strong constitutive expression (within L40) of
   - NLS (nuclear localisation sequence): from SV40 large T antigen
   - B42 ORF
   - HA-epitope: for anti-HA (hemagglutinin) antibodies
   - MCS: 2 unique restriction sites: EcoRI, XhoI
   - ADH1 transcription termination: stabilises mRNA
- 2µ origin: replication of yeast plasmid (high copy)
- TRP1 gene: Auxotrophy selection in trp^- hosts (like L40)
- Ampicillin resistence gene: Selection in E. coli
- ColE1 origin: replication of bacteria plasmid (E. coli)

Media

For more media and stock solutions see at Standard Reagents for Molecular Biology

YC	Storage at 4 °C
1.2 g	YNB (Yeast Nitrogen Base, w/o ammoniumn sulfate and aminoacids)
20.0 g	glucose
5.0 g	(NH₄)₂SO₄
10.0 g	succinate
6.0 g	NaOH
1.25 g	specific aminoacid mixture (0.1 g and 0.05 g of each aminoacid, respectively)
	for agarplates: add ~ 15 g agar
	adjust vol. to 1,000 ml and autoclave
	pour agar plates after cooling down to ~ 50 ºC to avoid condensed water add antibiotics etc. only after autoclaving when cooled down
2 ml Zeocin (100 mg/ml)	for YC Z200 plates (=> 200 µg/ml)
2 ml 3-AT (1 M)	for YC -H 3AT plates (=> 200 µg/ml)

Zeo stock	Storage at -20 °C
100.0 mg/ml	zeocin in A. dest. (sold steril)
	add 25 µl to 100 ml low salt-LB => 25 µg/ml add 200 µl to 100 ml YC => 200 µg/ml

1 M 3-AT stock	Storage at 4 °C
1,68 g	3-amino-1,2,4-triazole in 20 ml A. dest.
	sterilise by filtration (0.2 µm PES)
	add 0.5 ml to 100 ml YC -H => 5 mM

aminoacid mixtures	Storage at RT
YC -WHUK	1.0 g each: Ade, Arg, Cys, Leu, Thr	0.5 g each: Asp, Ile, Met, Phe, Pro, Ser, Tyr, Val
YC -HUK	1.0 g each: Ade, Arg, Cys, Leu, Thr, Trp	0.5 g each: Asp, Ile, Met, Phe, Pro, Ser, Tyr, Val
YC -WU	1.0 g each: Ade, Arg, Cys, Leu, Thr, Lys	0.5 g each: Asp, Ile, Met, Phe, Pro, Ser, Tyr, Val, His
YC -W	1.0 g each: Ade, Arg, Cys, Leu, Thr, Lys, Ura	0.5 g each: Asp, Ile, Met, Phe, Pro, Ser, Tyr, Val, His
YC	1.0 g each: Ade, Arg, Cys, Leu, Thr, Lys, Trp, Ura	0.5 g each: Asp, Ile, Met, Phe, Pro, Ser, Tyr, Val, His

Media usage	Saccharomyces cerevisiae growths at 30 °C and needs about 2 days to grow up.
YC	Growth of Sc.c. strain L40 (without selection pressure)
YC Z200	Growth of Sc.c. L40 with pHL-vector (selection pressure for pHL)
YC -HUK Z200	Test of Sc.c. L40 with pHL-vector for autoactivation (selection pressure for pHL and LexA activation)
YC -W	Growth of Sc.c. L40 with pYT or pJG-vectors (selection pressure for pYT/pJG)
YC -WHUK	Test of Sc.c. L40 with pHL-vector for autoactivation (selection pressure for pYT/pJG and LexA activation)
YC -W Z200	Growth of Sc.c. L40 with pYT/pJG- and pHL-vectors (selection pressure for both pYT/pJG and pHL)
YC -WHUK Z200	Test of Sc.c. L40 with pYT/pJG- and pHL-vector for protein-protein interactions (selection pressure for pYT/pJG and pHL and LexA activation)
YC -WHUK Z200 3AT	Test of Sc.c. L40 with pYT/pJG- and pHL-vector for strong protein-protein interactions (selection pressure for pYT/pJG and pHL and LexA activation). The LexA-activation results in His-prototrophy and 3-AT is inhibiting this His-synthesis, thus strong protein-protein interactions are needed to overcome this.

PBS	Storage at RT
1.78 g	Na₂HPO₄ * 2 H₂O (0.01 M)
14.61 g	NaCl (0.25 M)
	adjust vol. to 1000 ml with A. dest. and autoclave

Yeast transformation

Introducing the hybrid vector(s) into the yeast cells. Vectors bases on the 2-µm DNA are maintained stable and in high copy number (50 - 100) within the cells.

10x LiOAc (1 M) Storage at RT

10.2 g Li-acetate (=> 1 M)

90 ml A. dest

adjust with HAc to pH 7.5

adjust vol. to 100 ml with A. dest. and autoclave

LiOAc/TE Storage at RT

10 ml 10x LiOAc

10 ml 10x TE-buffer

adjust vol. to 100 ml with A. dest. and autoclave

LiOAc/PEG/TE Storage at RT

20 ml 10x LiOAc

20 ml 10x TE-buffer

100 g PEG 3350

adjust vol. to 200 ml with A. dest. and autoclave
Keep bottle tightly closed to prevent evaporation

salmon DNA (2 µg/µl) Storage at -20 °C

20 mg salmon sperm DNA (herring sperm DNA can also be used)

10 ml TE-buffer

dissolve by stirring overnight at 4 °C
boil for 5 min and chill on ice afterwards
aliquot a 500 µl portions and congelate

1 M Mannitol Storage at 4 °C

9,1 g mannitol

50 ml A. dest.

sterilise by autoclaving

YC/Mannitol Storage at 4 °C

9,1 g mannitol

50 ml YC-medium

sterilise by autoclaving

Heat shock

Yeast cells are exposed to a thermic shock (42 °C), which allows the entrance of small DNA

Inoculate colonies from selective YC plates in a test tube with 2 ml of the same YC medium. Let the cells grow for 1 day 30 °C.
- Next day inoculate 3.5 ml (prewarmed) YC selective medium with the 0.5 ml of the grown culture and let them grow up again to a OD₆₀₀ ~ 1 (about 5 h).
- Transfer cells into a 2 ml Eppendorf tube and centrifuge 1 min at 6,000 g. Discharge supernatant and repeat this step for the rest of the cells
- Resuspend cells in 2 ml PBS, centrifuge (1 min, 6,000 g) and discharge supernatant
- Resuspend cells in 2 ml TE/LiOAc, centrifuge (1 min, 6,000 g) and discharge supernatant
- Resuspend cells in 250 µl TE/LiOAc and incubate cells for 15 min at 30 °C
- Meanwhile prepare the transformation mix: join 300 µl LiOAc/PEG/TE-buffer, 20 µl salmon DNA, 2-5 µg (= 2-5 µl vector from a Midi-prep) and vortex vigorously
- Centrifuge cells (1 min, 6000 g), discharge supernatant and add the transformation mix. Mix cells by pipetting
- Incubate cells for 30 min at 30 °C, add 20 µl DMSO and incubate for 20 min at 42 °C. Shake cells to avoid sedimentation
- Chill cells on ice for 1 min
- Centrifuge cells (1 min, 6,000 g), discharge supernatant and add 1 ml YC medium
- Incubate cells 2-3 h at 30°C (shaking)
- Plate 10 µl, 100 µl and rest on selective YC agarplates
- Incubate plates for 2 - 7 days at 30 °C

Electroporation

Inoculate colonies from selective YC plates in a test tube with 2 ml of the same YC medium. Let the cells grow for 1 day 30 °C.
- Next day inoculate 3.5 ml (prewarmed) YC selective medium with the 0.5 ml of the grown culture and let them grow up again to a OD₆₀₀ ~ 1 (about 5 h).
- Transfer cells into a 2 ml Eppendorf tube and centrifuge 1 min at 6,000 g. Discharge supernatant and repeat this step for the rest of the cells
- Resuspend cells in 2 ml cold PBS, centrifuge (1 min, 6,000 g) and discharge supernatant
- Resuspend cells in 2 ml ice cold mannitol
- Add 20 µl DTT and incubate for 10 min at RT
- Centrifuge cells (1 min, 6,000 g), discharge supernatant
- Resuspend cells in 50 µl ice cold mannitol
- Add 2-5 µg plasmid DNA (= 2-5 µl vector from a Midi-prep) to the cells. Mix by softly pipetting (cutted tip) and
- Transfer cells into a cold electroporation cuvette and pulse at 7.500 V/cm (for a 10 mm gap: 750 V)
- Add immediately 1 ml YC/Mannitol and incubate cells for 2-3 h at 30 °C (shaking)
- Plate 10 µl, 100 µl and rest on selective YC agarplates
- Incubate plates for 2 - 7 days at 30 °C

β-Galactosidase Assay

The aim is to quantificate the searched protein-protein interaction: the stronger the interactions, the better they act as the transcription factor for the synthesis of the β-galactosidase, the more coloured product can be formed within a time.

The principle of the test is that the β-galactosidase hydrolysis the colourless ONPG forming galactose and yellow o-nitrophenol, with absorbs light at 420 nm.
ONPG

Z-buffer	Storage at RT
10.68 g	Na₂HPO₄ * 2 H₂O (60 mM)
6.24 g	NaH₂PO₄ * 2 H₂O (40 mM)
0.75 g	KCl (10 mM)
0.246 g	MgSO₄ * 7 H₂O (1 mM)
	dissolve in 900 ml A. dest. and adjust pH to 7.0
	adjust vol. to 1000 ml with A. dest. and autoclave

Z-buffer/β-ME	prepare daily fresh (50 reactions)
35 ml	Z-buffer
95 µl	β-mercaptoethanol (T)

ONPG solution	prepare daily fresh (50 reactions)
32 mg	o-nitrophenyl-β-D-galactopyranoside
8 ml	Z-buffer (=> 4 mg/ml)

1 M Na₂CO₃	prepare daily fresh (50 reactions)
2.12 g	Na₂CO₃ (Xi)
	adjust vol. to 20 ml with A. dest.

100 mM PMSF (T, F)	Storage at -20 °C in 100 µl aliquots
17.4 mg	PMSF, Phenylmethylsulfonyl fluoride (T)
1 ml	Isopropanol (F)
	PMSF, a protease inhibitor, degrades rapidly in water or is inactivated by DTT or β-ME, thus add PMSF right before the cell disruption. A less toxic alternative is AEBSF (4-[2-Aminoethyl]-benzensulfonylfluorid)

Inoculate colonies from YC -WHUK Z200 or better YC -WHUK Z200 3AT plates in a test tube with 2 ml YC -W Z200 or YC -WHUK Z200. Let the cells grow for 1 day 30 °C.
- Next day inoculate 1.75 ml (prewarmed) YC -W Z200 or YC -WHUK Z200 with 250 µl grown culture and let them grow up again to a OD₆₀₀ ~ 1 (about 5 h).
  Do not overgrow cells, as formed ethanol may act as an inhibitor (for [full length] ADH-promotor)
- Vortex the tube and determine the exact OD₆₀₀ for each test tube (100 µl cells 900 µl PBS, use PBS as blank).
  Keep the cells meanwhile on ice.
- Transfer 1.5 ml cell culture into a Eppendorf tube and centrifuge 1 min at 10,000 g
- Resuspend the cells in 1 ml ice cold PBS and centrifuge 1 min at 10,000 g
- Resuspend cells in 100 µl Z-buffer. Use 3 blanks with Z-buffer only
A) Liquid Nitrogen:
  - disrupt cells by 3 freeze (liquid nitrogen) - thawn (37 °C) cycles.
    Point into the cap of the Eppendorf tube a small hole to avoid popping off the cap.
B) Toluene (when liquid nitrogen is not accessible):
  - add 5 µl toluene (F, Xn) [and 10 µl PMSF solution (?)]
  - vortex 6 times for 15 s at top speed [with glass beads ?]
- Add 700 µl Z-buffer/β-ME
- Add 160 µl ONPG solution, mix and start the time
  Start each sample/test with a 10 s time shift for determining the exact time
- Incubate mixture at 37 °C untill formation of a yellow colour
- If so, stop the reaction by adding 400 µl Na₂CO₃ and take the time. Stop also one blank
- Otherwise stop the reactions after 1 h
- Centrifuge the mixture for 3 min at 12.000 g
- Determine the absorption at 420 nm. Do not mix up the sedimented cells
- Calculate β-galactosidase activity:

rel. unit = (A₄₂₀ * 1000) / (t_[min] * 1.5 * OD₆₀₀)

Last modified: 25.09.2009

10x LiOAc (1 M)	Storage at RT
10.2 g	Li-acetate (=> 1 M)
90 ml	A. dest
	adjust with HAc to pH 7.5
	adjust vol. to 100 ml with A. dest. and autoclave

LiOAc/TE	Storage at RT
10 ml	10x LiOAc
10 ml	10x TE-buffer
	adjust vol. to 100 ml with A. dest. and autoclave

LiOAc/PEG/TE	Storage at RT
20 ml	10x LiOAc
20 ml	10x TE-buffer
100 g	PEG 3350
	adjust vol. to 200 ml with A. dest. and autoclave Keep bottle tightly closed to prevent evaporation

salmon DNA (2 µg/µl)	Storage at -20 °C
20 mg	salmon sperm DNA (herring sperm DNA can also be used)
10 ml	TE-buffer
	dissolve by stirring overnight at 4 °C boil for 5 min and chill on ice afterwards aliquot a 500 µl portions and congelate

1 M Mannitol	Storage at 4 °C
9,1 g	mannitol
50 ml	A. dest.
	sterilise by autoclaving

YC/Mannitol	Storage at 4 °C
9,1 g	mannitol
50 ml	YC-medium
	sterilise by autoclaving