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

HybriZAP Vector System

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 Two-Hybrid Systemfor 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

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......

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

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

Questions?