Transcript HybriZAP Two-Hybrid Vector System

Two-Hybrid Vector Systems
Detection of Protein-Protein
Interactions
Protein-Protein Interactions
• Essential role in most biological
processes
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Signal transduction
Cell growth and differentiation
Gene expression
Secretion
Metabolishm
DNA replication
Transcription / translation / splictin
Cell cycle control
Functional Genomics
• Protein kinases ( >2000); protein
phosphatases (>1000); receptors;
transcriptional regulators; signal
transducers; etc....
• Signaling molecules play pivotal roles
• Poor in vitro and in vivo function
correlation's
Protein-Protein Interaction Methods
• Traditional biochemical methods
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Co-immunoprecipitation
Protein crosslinking
Co-purification
Protein expression libraries
• Two-Hybrid System
– Protein folding and phosphorylation
– No protein purification
– Links genotype to phenotype
Two-Hybrid Applications
• Identify novel interacting proteins
• Verify interactions determined by
traditional methods
• Drug screening
• Functional mapping
– domains / amino acids
– post-translational modifications required
Two-Hybrid Systems
• Nuclear Yeast Systems
– GAL4
– LexA
• Membrane Yeast Systems
– CytoTrap ….Sos recruitment system (SRS)
Basics of Two-Hybrid Systems
• cDNA vector
– expresses random primed or oligo d(T) primed cDNA
– expressed protein is the “PREY” or “Target” protein
fused to a tranacriptional activation domain (GAL4 /
LexA systems) or is myrstilated (Stratagene’s
CytoTrap system)
• Bait vector
– expresses a known protein
– expressed protein is chimeric, part is the known bait
protein and part is a transcription factor DNA binding
domain (GAL4 / Lex A systems), or hSos (CytoTrap)
Nucleus
2. Target-AD hybrid
protein binds to bait
1. Bait-DBD hybrid protein
binds to DNA
Bait
DBD
Target
AD
3. Activation domain
switches on
transcription of
reporter gene(s)
HIS3
DNA Binding
Site
or
b-gal
4. Yeast cells expressing
target proteins that bind
to bait grow on media
lacking histidine and turn
blue in the presence of X-gal
GAL4 / LexA Considerations
• Expression of hybrid proteins and
localization to the nucleus
• GAL4 fusion partner must not interfere
with interactions
• Neither hybrid can activate transcription
of reporter gene without the other hybrid
protein
Limitations of GAL4 / LexA
• Bait proteins which activate transcription
of reporter genes in the absence of an
interaction target protein can not be used.
• Interactions must take place in the
nucleus
HybriZAP Vector System
Advantages of l AD Vector
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Cloning efficiency is higher
Background is lower
No size bias with l (0 ~ 6 kb inserts)
Pre-screening and screening for additional
clones with traditional methods are easier
• cDNA can be cloned directionally or nondirectional and random
• In vivo excision to plasmid libraries is simple
and fast
HybriZAP EXCISION PROCESS
HybriZAP
XL1-Blue
HybriZAP
Packaged
pAD-GAL4
Phagemid
XLOLR
Ex-Assist
Helper Phage
F pilus
Ex-Assist
Helper Phage
F pilus
a) Co-infection of XL1-Blue,
SupE E.coli cells
containing F’ episome
coding for F pilus and
bearing LamB receptors.
b) Excision Process.
HybriZAP, packaged pAD-GAL4
and packaged
Helper Phage are produced.
c) Infection of XLOLR,
Su-, lr , F’ E.coli cells.
Lambda phage cannot infect
and Ex-Assist Helper Phage
cannot replicate.
d) Plate on Ampicillian
plates. Resulting colonies
contain pAD-GAL4 phagemid
and insert DNA.
Yeast YRG-2 Reporter Genes
• LacZ
• Histidine
– GLA4 17 mer UAS
– Sensitive
– Easy and rapid assay
• Filter lift
• Quantitative liquid assay
– Not selectable
Endogenous GAL4 not expressed
GAL 80 is mutated
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GAL1 UAS
Very sensitive
Selectable
Not quantitative
Frozen Competent Yeast Cells
• thaw cells
• add plasmid DNA
• 30 minutes at 30oC, heat shock at 42oC for
8 min.
• add SD broth, grow 1 hour at 30oC
• pellet cells, resuspend, plate (3-7 days to
get colonies)
Stratagene’s GAL4 System
• HybriZAP is l or
plasmid based
• Directional libraries,
random nondirectional
• Reporter genes
integrated into YRG-2
• Vector kits, premade
and custom cDNA
libraries
• Power of l, ease of
plasmid
• Increased relative
size of the library
• Available as high
efficiency frozen cells
• Flexible options
/priced competitively
Two-Hybrid Products (GAL4)
• Complete HybriZAP 2.1 l kit
– Digested l vector, Gigapack, competent E. coli, cDNA
synthesis kit, control plasmids
• Complete plasmid kit
– Digested pAD-GAL4-2.1 vector, XL10-Gold
Ultracompetent E. coli, cDNA synthesis kit, control
plasmids, competent YRG-2 yeast
• Custom and Premade libraries
• Accessory kits
CytoTrap Two-Hybrid System
for the Detection of
Protein-Protein Interactions
CytoTrap customer
• Is beginning a two-hybrid screen and isn’t
sure if the bait needs glycosylation
• Has wanted to screen with a protein that
is a transcriptional activator or
inhibitor....maybe a PathDetect customer?
• Has been unsuccessful in a traditional
two-hybrid screen
Ras Activation
Ras
GTP
Ras
GDP
P
P
SOS
Greb2
In yeast, the Cdc25 protein has the
same function as human SOS.
Thus, hSOS can complement the
yeast Cdc25 protein…. but if you
remove the Greb-2 domain of
SOS, it can not localize to the
membrane to activate the Ras
pathway
CDC25H-2 Yeast Host
• Genotype:
MAT a, ura3, lys2, leu2, trp1, cdc25-2,
his3D200, ade, Gal+
• Phenotype:
Cdc25-2 yeast homologue of hSOS and
is a guanyl nucleotide exchange factor
(GEF)
• cdc25-2 protein is thermostable at 25oC
• cdc25-2 protein is not thermostable at 37oC
1. Target protein
becomes anchored
to cell membrane
Myristylation
Signal
Target
Cell Membrane
GDP
RAS
Bait
hSOS
2. Bait protein binds to target,
localizing SOS to membrane
GTP
3. SOS activates RAS by promoting
GDP/GTP exchange
4. RAS activates signaling cascade
that permits mutant yeast
CDC25H-2 to grow at 37oC
plasmid vector
5.6 kb
U
S
pMyr
EcoRI
Srf I/I
Sma
Xho I
Sal I
Bait vector
11.3 kb
S
pSOS
BamH I
Nco I
Srf I
Aat I
Sal I
Mlu I
BssH II
Sac I
Not I
Sac II
2× Pac I
Co-transform
pADH-Sos Gal 4 AD Bait
pGal1-Myr yeast cDNA library
Select on -UL/Glu Plates @ 25oC
2 days
Replica Plate on -UL/Gal Plates @37oC
Patch “positive colonies”
-UL/Glu
25oC 37oC
No Interaction:
Temp Revert:
Interaction:
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-UL/Gal
25oC
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+
37oC
+
344+
53 “positive” interaction clones selected
Isolate pGal1 Myr yeast cDNA plasmid DNA
Co-transform
-UL/Gal
25oC 37oC
pSOS GAL4:
pSOS:
+
+
+
-
Putative Positive
Further Analysis
CytoTrap products:
• CytoTrap XR Library Construction Kit
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cDNA synthesis kit
pMyr XR vector (Xho I and EcoRI digested)
XL10-Gold ultracompetent cells
pSos vector
XL1-Blue subcloning competent cells
cdc25H yeast strain
CytoTrap products:
• CytoTrap Vector Kit
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pMyr vector (supercoiled)
pSos vector (supercoiled)
2 positive control plasmids
2 negative control plasmids
cdc25H yeast strain
pMyr XR vector
pMyr vector (supercoiled)
pSos vector
cdc25H yeast strain
In Conclusion
• Directional cloning / or random Eco RI cloning of
cDNA
• Exceptional premades available now
• Brochure available
• The CytoTrap system is the first is transcriptional
activation independent yeast two-hybrid system
Competition
• Remember....CytoTrap is a completely
different way to look for protein-protein
interactions
• Clontech Matchmaker systems
– GAL4 and LexA
• Invitrogen Hybrid Hunter
– LexA
• Other small companies......
Mammalian Two-Hybrid System
More tools for functional genomics
Why Mammalian?
• Verification of mammalian interactions
discovered in yeast
• Proteins more likely to have native
conformation
• Results more likely to represent
biologically significant interactions
• Yeast do not phosphorylate tyrosine
Nucleus
Bait (X) Target (Y)
NF-kB AD
Gal4 BD
pReporter
Gal4
Gal4
Gal4
Gal4
Gal4
Figure 1
TATA
Reporter Gene
Why Nf-kB activation domain?
pGal4BD NF-kB # 478
Gal4 (1-147)
NF-kB (283-550)
pGal4BD NF-kB # 476
Gal4 (1-147)
NF-kB (364-550)
pGal4BD NF-kB # 479
Gal4 (1-147)
NF-kB (519-550)
pGal4BD VP16
Gal4 (1-147)
VP16 (411-455)
Luciferase Activity (RLU)
18000000
16000000
14000000
12000000
10000000
8000000
6000000
4000000
2000000
0
Gal4BD-VP16
Gal4BD-NFkB 476
Gal4BD-NFkB 479
Gal4BD-NFkB 478
CHO
293
HeLa-luc
CMV promoter
ColE1 ori
pAD
NF-kB
4141 bps
MCS
AmpR
SV40 poly(A)
LoxP
f1-ori
BamHI
SrfI
EcoRI
HindIII
NotI
Eco52I
SalI
XbaI
XhoI
CMV promoter
Col E1 replication ori
GAL4BD
TK poly(A) signal
MCS
pBD
4598 bps
SV40 poly (A)
Neo/Kan
f1(-) ori
SV40 early promoter
BamHI
NheI
SrfI
SmaI
XmaI
EcoRI
HindIII
NotI
AccI
SalI
XbaI
PstI
Ecl136II
SacI
Acc65I
KpnI
BglII
HLR cell line
30000
25000
20000
15000
10000
5000
pBD-53 +
pAD-TRAF2
pBD-CD40 +
pAD-SV40 T
pBD-53 +
pAD-TRAF2
pBD-CD40
+ pAD-SV40 T
COS
2500000
2000000
1500000
1000000
500000
0
pBD-53 +
pAD-TRAF2
pBD-CD40
+ pAD-SV40 T
pBD-53 +
pAD-SV40 T
Figure 4
293
2500000
2000000
1500000
1000000
500000
0
pBD-53 +
pAD-SV40 T
Luciferase Activity)
0
pBD-53 +
pAD-SV40 T
Luciferase Activity
0
CHO
14000000
12000000
10000000
8000000
6000000
4000000
2000000
Luciferase Activity
Luciferase Activity
Luciferase Activity (
Interaction between P53 and SV40T results in activation of
luciferase activity
pBD-53 +
pAD-TRAF2
pBD-CD40 +
pAD-SV40 T
pBD-53 +
pAD-SV40 T
HeLa
60000
50000
40000
30000
20000
10000
0
pBD-53 +
pAD-TRAF2
pBD-CD40
+ pAD-SV40 T
pBD-53 +
pAD-SV40 T
A
Using different reporters
SEAP
SEAP Units
250000
200000
150000
100000
50000
0
pBD 53 + pAD
TRAF2
pBD CD40 +
pAD SV40 T
pBD 53 + pAD
SV40 T
b-galactosidase
Units
b-Gal
C
1.2
1
0.8
0.6
0.4
0.2
0
pBD 53 + pAD
TRAF2
pBD CD40 +
pAD SV40 T
Figure 5
pBD 53 + pAD
SV40 T
Competition
• Clontech
– VP16 Activation Domain / CAT reporter
• Promega CheckMate Mammalian TwoHybrid System
– VP16 Activation Domain / dual Renilla and firefly
Luciferase reporter
• Stratagene Advantages
– Increased activity of Nf-kB activator
– Multiple reporter options
Questions?