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Two-Hybrid Vector Systems Detection of Protein-Protein Interactions.
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Transcript of Two-Hybrid Vector Systems Detection of Protein-Protein Interactions.
Protein-Protein Interactions
• Essential role in most biological processes– 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– 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)
HIS3
-galorDNA Binding
Site
Nucleus
1. Bait-DBD hybrid protein binds to DNA
BaitDBD
Target AD
2. Target-AD hybrid protein binds to bait
3. Activation domain switches on transcription of reporter gene(s)
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
Advantages of AD Vector
• Cloning efficiency is higher• Background is lower• No size bias with (0 ~ 6 kb inserts)• Pre-screening and screening for additional clones
with traditional methods are easier• cDNA can be cloned directionally or non-
directional and random• In vivo excision to plasmid libraries is simple and
fast
a) Co-infection of XL1-Blue,SupE E.coli cellscontaining F’ episome coding for F pilus and bearing LamB receptors.
Ex-Assist Helper Phage
HybriZAP
b) Excision Process. HybriZAP, packaged pAD-GAL4and packaged Helper Phage are produced.HybriZAP
Ex-Assist Helper Phage
Packaged pAD-GAL4Phagemid
F pilusXL1-Blue
c) Infection of XLOLR, Su-, r , F’ E.coli cells. Lambda phage cannot infect and Ex-Assist Helper Phagecannot replicate.
F pilusXLOLR
d) Plate on Ampicillianplates. Resulting coloniescontain pAD-GAL4 phagemid and insert DNA.
HybriZAP EXCISION PROCESS
Yeast YRG-2 Reporter Genes
• LacZ– GLA4 17 mer UAS
– Sensitive
– Easy and rapid assay• Filter lift
• Quantitative liquid assay
– Not selectable
• Histidine– GAL1 UAS
– Very sensitive
– Selectable
– Not quantitative
Endogenous GAL4 not expressedGAL 80 is mutated
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 or plasmid based
• Directional libraries, random non-directional
• Reporter genes integrated into YRG-2
• Vector kits, premade and custom cDNA libraries
• Power of , 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 kit– Digestedvector, 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 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
P
P
SOS
Greb2
Ras
GDP
RasGTP
In yeast, the Cdc25 protein has thesame function as human SOS. Thus, hSOS can complement the yeast Cdc25 protein…. but if youremove the Greb-2 domain of SOS, it can not localize to themembrane to activate the Ras pathway
CDC25H-2 Yeast Host
• Genotype: , ura3, lys2, leu2, trp1, cdc25-2, his3200, ade, Gal+
• Phenotype:Cdc25-2 yeast homologue of hSOS and
is a guanyl nucleotide exchange factor (GEF)
• cdc25-2 protein is thermostable at 25C• cdc25-2 protein is not thermostable at 37oC
Cell Membrane
1. Target protein becomes anchored to cell membrane
Target
Myristylation Signal
2. Bait protein binds to target, localizing SOS to membrane
Bait
hSOS
RASGDP
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
URA
3
pMyrplasmid vector
5.6 kb
ColE1 or
igin
RII/II
I
Eco Srf Sma Xho Sal
Myr
MCS
2 or
i PGAL l
f1 origin
TCYC1
Cam r
LEU
2
pSOSBait vector
11.3 kb
ColE1 or
igi
n
2 ori H II
IIII
H IIIIII
2×
BamNco Srf Aat Sal Mlu BssSac Not Sac
IPac
hSOS
T AD
HIM
CS
PADH
Amp r
Co-transform pADH-Sos Gal 4 AD Bait
pGal1-Myr yeast cDNA library
Select on -UL/Glu Plates @ 25oC2 days
Replica Plate on -UL/Gal Plates @37oC
-UL/Glu -UL/Gal 25oC 37oC 25oC 37oC
No Interaction: + - + -Temp Revert: + + + +Interaction: + - + 344+
Patch “positive colonies”
53 “positive” interaction clones selected
Isolate pGal1 Myr yeast cDNA plasmid DNA
Co-transform
-UL/Gal
25oC 37oC
pSOS GAL4: + + pSOS: + -
Putative PositiveFurther Analysis
CytoTrap products:
• CytoTrap XR Library Construction Kit– 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
– 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
• 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
In Conclusion
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......
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
Gal4 Gal4 Gal4 Gal4 Gal4 TATA Reporter Gene
Gal4 BD
Bait (X) NF-B ADTarget (Y)
pReporter
Nucleus
Figure 1
pGal4BD NF-B # 478
pGal4BD NF-B # 476
pGal4BD NF-B # 479
pGal4BD VP16
Gal4 (1-147) NF-B (283-550)
VP16 (411-455)
NF-B (364-550)
NF-B (519-550)Gal4 (1-147)
Gal4 (1-147)
Gal4 (1-147)
Why Nf-B activation domain?
0
2000000
4000000
6000000
8000000
10000000
12000000
14000000
16000000
18000000
CHO 293 HeLa-luc
Lu
cife
rase
Act
ivit
y (R
LU
)
Gal4BD-VP16
Gal4BD-NFB 476
Gal4BD-NFB 479
Gal4BD-NFB 478
pAD
4141 bps BamHISrfIEcoRIHindIIINotIEco52ISalIXbaIXhoI
CMV promoter
NF-kB
MCS
SV40 poly(A)
f1-oriLoxP
AmpR
ColE1 ori
pBD
4598 bps
BamHINheISrfISmaIXmaIEcoRIHindIIINotIAccISalIXbaIPstIEcl136IISacIAcc65IKpnIBglII
CMV promoter
GAL4BD
MCS
SV40 poly (A)
f1(-) ori
SV40 early promoter
Neo/Kan
TK poly(A) signal
Col E1 replication ori
0100002000030000400005000060000
pBD-53 +pAD-TRAF2
pBD-CD40+ pAD-SV40 T
pBD-53 +pAD-SV40 T
HeLa
02000000400000060000008000000
100000001200000014000000
pBD-53 +pAD-TRAF2
pBD-CD40+ pAD-SV40 T
pBD-53 +pAD-SV40 T
CHO
0
500000
1000000
1500000
2000000
2500000
pBD-53 +pAD-TRAF2
pBD-CD40+ pAD-SV40 T
pBD-53 +pAD-SV40 T
COS
Luc
ifer
ase
Act
ivit
y
Luc
ifer
ase
Act
ivit
y)
Luc
ifer
ase
Act
ivit
y
05000
1000015000200002500030000
pBD-53 +pAD-TRAF2
pBD-CD40 +pAD-SV40 T
pBD-53 +pAD-SV40 T
HLR cell line
Luc
ifer
ase
Act
ivit
y (
0
500000
1000000
1500000
2000000
2500000
pBD-53 +pAD-TRAF2
pBD-CD40 +pAD-SV40 T
pBD-53 +pAD-SV40 T
293
Luc
ifer
ase
Act
ivit
y
Interaction between P53 and SV40T results in activation of luciferase activity
Figure 4
A
0
0.2
0.4
0.6
0.8
1
1.2
pBD 53 + pADTRAF2
pBD CD40 +pAD SV40 T
pBD 53 + pADSV40 T
C
0
50000
100000
150000
200000
250000
pBD 53 + pADTRAF2
pBD CD40 +pAD SV40 T
pBD 53 + pADSV40 T
Figure 5
SE
AP
Uni
ts-
gala
ctos
idas
e U
nits
Using different reportersSEAP
-Gal
Competition
• Clontech– VP16 Activation Domain / CAT reporter
• Promega CheckMate Mammalian Two-Hybrid System– VP16 Activation Domain / dual Renilla and firefly
Luciferase reporter
• Stratagene Advantages – Increased activity of Nf-kB activator– Multiple reporter options