Interaction Trap/Two‐Hybrid System to Identify Interacting Proteins

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Abstract
Table of Contents
Materials
Figures
Literature Cited

Abstract

The yeast two?hybrid method (or interaction trap) is a powerful technique for detecting protein interactions. The procedure is performed using transcriptional activation of a dual reporter system in yeast to identify interactions between a protein of interest (the bait protein) and the candidate proteins for interaction. The method can be used to screen a protein library for interactions with a bait protein or to test for association between proteins that are expected to interact based on prior evidence. Interaction mating facilitates the screening of a library with multiple bait proteins. Curr. Protoc. Neurosci. 55:4.4.1?4.4.35. © 2011 by John Wiley & Sons, Inc.

Keywords: protein interactions; yeast two?hybrid; interaction trap; interaction mating

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Introduction
Basic Protocol 1: Producing and Characterizing a Bait Strain
Alternate Protocol 1: Confirmation of Fusion Protein Synthesis by Repression Assay
Basic Protocol 2: Performing an Interactor Hunt
Alternate Protocol 2: Performing a Hunt by Interaction Mating
Support Protocol 1: Preparation of Sheared Salmon Sperm Carrier DNA
Support Protocol 2: Yeast Colony Hybridization
Support Protocol 3: Microplate Plasmid Rescue
Reagents and Solutions
Commentary
Figures
Tables

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Materials

Basic Protocol 1: Producing and Characterizing a Bait Strain

Materials

DNA encoding the protein of interest
Plasmids (see Table 4.4.1 ): e.g., pEG202 (Fig. ), pSH18‐34 (Fig. ), pSH17‐4, pRFHM1
Yeast strain: e.g., EGY48 (ura3 trp1 his3 3LexA ‐operator‐LEU2 ; see Table 4.4.2 )
100‐mm complete minimal (CM) medium dropout plates (Treco and Lundblad, ) with 2% (w/v) glucose (Glu) or 2% (w/v) galactose (Gal):
- Glu/CM –Ura –His
- Gal/CM –Ura –His
- Gal/CM –Ura –His –Leu
Glu/CM Xgal and Gal/CM Xgal plates (Treco and Lundblad, )
CM dropout liquid medium (Treco and Lundblad, ) with 2% (w/v/) glucose: Glu/CM −Ura −His
2× Laemmli sample buffer (see recipe )
Antibody to LexA or fusion domain
H₂ O, sterile

30°C incubator
100°C water bath

Additional reagents and equipment for subcloning (Struhl, ), lithium acetate transformation of yeast (Becker and Lundblad, ), filter lift or liquid assay for β‐galactosidase (Reynolds et al., ), SDS‐PAGE (Gallagher, ), and immunoblotting (Gallagher et al., )

Table 4.4.1 Materials Interaction Trap Plasmids a a ^, b b Interaction Trap Plasmids Interaction Trap Yeast Selection Strains f Interaction Trap Yeast Selection Strains

	Selection
Plasmid name/source	In yeast	In E. coli	Comment/description
LexA fusion plasmids
pEG202 c ^, d	HIS3	Ap^r	Contains an ADH promoter that expresses LexA followed by polylinker
pJK202	HIS3	Ap^r	Like pEG202, but incorporates nuclear localization sequences between LexA and polylinker; used to enhance translocation of bait to nucleus
pNLexA d	HIS3	Ap^r	Contains an ADH promoter that expresses polylinker followed by LexA for use with baits where their amino‐terminal residues must remain unblocked
pGilda d	HIS3	Ap^r	Contains a GAL1 promoter that expresses same LexA and polylinker cassette as pEG202 for use with baits where their continuous presence is toxic to yeast
pEE202I	HIS3	Ap^r	An integrating form of pEG202 that can be targeted into HIS3 following digestion with Kpn I; for use where physiological screen requires lower levels of bait to be expressed
pRFHM1 d (control)	HIS3	Ap^r	Contains an ADH promoter that expresses LexA fused to the homeodomain of bicoid to produce nonactivating fusion used; as positive control for repression assay, negative control for activation and interaction assays
pSH17‐4 d (control)	HIS3	Ap^r	ADH promoter expresses LexA fused to GAL4 activation domain; used as a positive control for transcriptional activation
pMW101 e	HIS3	Cm^r	Same as pEG202, but with altered antibiotic resistance markers; basic plasmid used for cloning bait
pMW103 e	HIS3	Km^r	Same as pEG202, but with altered antibiotic resistance markers; basic plasmid used for cloning bait
pHybLex/Zeo	Zeo^r	Zeo^r	Bait cloning vector compatible with interaction trap and all other two‐hybrid systems; minimal ADH promotor expresses LexA followed by extended polylinker
Activation domain fusion plasmids
pJG4‐5 c ^, d	TRP1	Ap^r	Contains a GAL1 promoter that expresses nuclear localization domain, transcriptional activation domain, HA epitope tag, cloning sites; used to express cDNA libraries
pJG4‐5I	TRP1	Ap^r	An integrating form of pJG4‐5 that can be targeted into TRP1 by digestion with Bsu 36I (New England Biolabs); to be used with pEE202I to study interactions that occur physiologically at low protein concentrations
pYESTrp	TRP1	Ap^r	Contains a GAL1 promoter that expresses nuclear localization domain, transcriptional activation domain, V5 epitope tag, multiple cloning sites; contains f1 ori and T7 promoter/flanking site used to express cDNA libraries (Invitrogen)
pMW102 e	TRP1	Km^r	Same as pJG4‐5, but with altered antibiotic resistance markers; no libraries yet available
pMW104 e	TRP1	Cm^r	Same as pJG4‐5, but with altered antibiotic resistance markers; no libraries yet available
LacZ reporter plasmids
pSH18‐34 d	URA3	Ap^r	Contains eight LexA operators that direct transcription of the lacZ gene; one of the most sensitive indicator plasmids for transcriptional activation
pJK103 d	URA3	Ap^r	Contains two LexA operators that direct transcription of the lacZ gene; an intermediate reporter plasmid for transcriptional activation
pRB1840 d	URA3	Ap^r	Contains one LexA operator that directs transcription of the lacZ gene; one of the most stringent reporters for transcriptional activation
pMW112 e	URA3	Km^r	Same as pSH18‐34, but with altered antibiotic resistance marker
pMW109 e	URA3	Km^r	Same as pJK103, but with altered antibiotic resistance marker
pMW111 e	URA3	Km^r	Same as pRB1840, but with altered antibiotic resistance marker
pMW107 e	URA3	Cm^r	Same as pSH18‐34, but with altered antibiotic resistance marker
pMW108 e	URA3	Cm^r	Same as pJK103, but with altered antibiotic resistance marker
pMW110 e	URA3	Cm^r	Same as pRB1840, but with altered antibiotic resistance marker
pJK101 d (control)	URA3	Ap^r	Contains a GAL1 upstream activating sequence followed by two LexA operators followed by lacZ gene; used in repression assay to assess bait binding to operator sequences
Strain	Relevant genotype	Number of operators	Comments/description
EGY48 g	MAT α trp1 , his3 , ura3 , lexAops‐LEU2	6	lexA operators direct transcription from the LEU2 gene; EGY48 is a basic strain used to select for interacting clones from a cDNA library
EGY191	MATα trp1 , his3 , ura3 , lexAops‐LEU2	2	EGY191 provides a more stringent selection than EGY48, producing lower background with baits with instrinsic ability to activate transcription
L40^b	MATα trpl , leu 2, ade 2, GAL4, lexAops‐HIS34 , lexAops‐lacZ8		Expression driven from GAL1 promoter is constitutive in L40 (inducible in EGY strains); selection is for HIS prototrophy. Integrated lacZ reporter is considerably less sensitive than pSH18‐34 maintained in EGY strains.

^a All plasmids contain a 2µm origin for maintenance in yeast, as well as a bacterial origin of replication, except where noted (pEE202I, pJG4‐5I).

^b Interaction Trap reagents represent the work of many contributors: the original basic reagents were developed in the Brent laboratory (Gyuris et al., ). Plasmids with altered antibiotic resistance markers (all pMW plasmids) were constructed at Glaxo in Research Triangle Park, N.C. (Watson et al., ). Plasmids and strains for specialized applications have been developed by the following individuals: E. Golemis, Fox Chase Cancer Center, Philadelphia, Pa. (pEG202); cumulative efforts of I. York, Dana‐Farber Cancer Center, Boston, Mass. and M. Sainz and S. Nottwehr, U. Oregon (pNLexA); D.A. Shaywitz, MIT Center for Cancer Research, Cambridge, Mass. (pGilda); R. Buckholz, Glaxo, Research Triangle Park, N.C. (pEE202I, pJG4‐5I); J. Gyuris, Mitotix, Cambridge, Mass. (pJG4‐5); R.L. Finley, Wayne State University School of Medicine, Detroit, Mich. (pSH17‐4 pRFHM1); S. Hanes, Wadsworth Institute, Albany, N.Y. (pSH17‐4, pSH18‐34); J. Kamens, BASF, Worcester, Mass. (pJK101, pJK103, pJK202); R. Brent, The Molecular Sciences Institute, Berkeley, Calif. (pRB1840).

^c Sequence data are available for pEG202 (pLexA), accession number U89960, and pJG4‐5, accession number U89961.

^d Plasmids and strains available from OriGene.

^e In pMW plasmids the ampicillin resistance gene (Ap^r ) is replaced with the chloramphenicol resistance gene (Cm^r ) and the kanamycin resistance gene (Km^r ) from pBC SK(+) and pBK‐CMV (Stratagene), respectively. The choice between Km^r and Cm^r or Ap^r plasmids is a matter of personal taste; use of basic Ap^r plasmids is described in the basic protocols. Use of the more recently developed reagents would facilitate the purification of library plasmid in later steps by eliminating the need for passage through KC8 bacteria, with substantial saving of time and effort. Ap^r has been maintained as marker of choice for the library plasmid because of the existence of multiple libraries already possessing this marker. These plasmids are the basic set of plasmids recommended for use.

Table 4.4.2 Materials Interaction Trap Plasmids a a ^, b b Interaction Trap Plasmids Interaction Trap Yeast Selection Strains f Interaction Trap Yeast Selection Strains

	Selection
Plasmid name/source	In yeast	In E. coli	Comment/description
LexA fusion plasmids
pEG202 c ^, d	HIS3	Ap^r	Contains an ADH promoter that expresses LexA followed by polylinker
pJK202	HIS3	Ap^r	Like pEG202, but incorporates nuclear localization sequences between LexA and polylinker; used to enhance translocation of bait to nucleus
pNLexA d	HIS3	Ap^r	Contains an ADH promoter that expresses polylinker followed by LexA for use with baits where their amino‐terminal residues must remain unblocked
pGilda d	HIS3	Ap^r	Contains a GAL1 promoter that expresses same LexA and polylinker cassette as pEG202 for use with baits where their continuous presence is toxic to yeast
pEE202I	HIS3	Ap^r	An integrating form of pEG202 that can be targeted into HIS3 following digestion with Kpn I; for use where physiological screen requires lower levels of bait to be expressed
pRFHM1 d (control)	HIS3	Ap^r	Contains an ADH promoter that expresses LexA fused to the homeodomain of bicoid to produce nonactivating fusion used; as positive control for repression assay, negative control for activation and interaction assays
pSH17‐4 d (control)	HIS3	Ap^r	ADH promoter expresses LexA fused to GAL4 activation domain; used as a positive control for transcriptional activation
pMW101 e	HIS3	Cm^r	Same as pEG202, but with altered antibiotic resistance markers; basic plasmid used for cloning bait
pMW103 e	HIS3	Km^r	Same as pEG202, but with altered antibiotic resistance markers; basic plasmid used for cloning bait
pHybLex/Zeo	Zeo^r	Zeo^r	Bait cloning vector compatible with interaction trap and all other two‐hybrid systems; minimal ADH promotor expresses LexA followed by extended polylinker
Activation domain fusion plasmids
pJG4‐5 c ^, d	TRP1	Ap^r	Contains a GAL1 promoter that expresses nuclear localization domain, transcriptional activation domain, HA epitope tag, cloning sites; used to express cDNA libraries
pJG4‐5I	TRP1	Ap^r	An integrating form of pJG4‐5 that can be targeted into TRP1 by digestion with Bsu 36I (New England Biolabs); to be used with pEE202I to study interactions that occur physiologically at low protein concentrations
pYESTrp	TRP1	Ap^r	Contains a GAL1 promoter that expresses nuclear localization domain, transcriptional activation domain, V5 epitope tag, multiple cloning sites; contains f1 ori and T7 promoter/flanking site used to express cDNA libraries (Invitrogen)
pMW102 e	TRP1	Km^r	Same as pJG4‐5, but with altered antibiotic resistance markers; no libraries yet available
pMW104 e	TRP1	Cm^r	Same as pJG4‐5, but with altered antibiotic resistance markers; no libraries yet available
LacZ reporter plasmids
pSH18‐34 d	URA3	Ap^r	Contains eight LexA operators that direct transcription of the lacZ gene; one of the most sensitive indicator plasmids for transcriptional activation
pJK103 d	URA3	Ap^r	Contains two LexA operators that direct transcription of the lacZ gene; an intermediate reporter plasmid for transcriptional activation
pRB1840 d	URA3	Ap^r	Contains one LexA operator that directs transcription of the lacZ gene; one of the most stringent reporters for transcriptional activation
pMW112 e	URA3	Km^r	Same as pSH18‐34, but with altered antibiotic resistance marker
pMW109 e	URA3	Km^r	Same as pJK103, but with altered antibiotic resistance marker
pMW111 e	URA3	Km^r	Same as pRB1840, but with altered antibiotic resistance marker
pMW107 e	URA3	Cm^r	Same as pSH18‐34, but with altered antibiotic resistance marker
pMW108 e	URA3	Cm^r	Same as pJK103, but with altered antibiotic resistance marker
pMW110 e	URA3	Cm^r	Same as pRB1840, but with altered antibiotic resistance marker
pJK101 d (control)	URA3	Ap^r	Contains a GAL1 upstream activating sequence followed by two LexA operators followed by lacZ gene; used in repression assay to assess bait binding to operator sequences
Strain	Relevant genotype	Number of operators	Comments/description
EGY48 g	MAT α trp1 , his3 , ura3 , lexAops‐LEU2	6	lexA operators direct transcription from the LEU2 gene; EGY48 is a basic strain used to select for interacting clones from a cDNA library
EGY191	MATα trp1 , his3 , ura3 , lexAops‐LEU2	2	EGY191 provides a more stringent selection than EGY48, producing lower background with baits with instrinsic ability to activate transcription
L40^b	MATα trpl , leu 2, ade 2, GAL4, lexAops‐HIS34 , lexAops‐lacZ8		Expression driven from GAL1 promoter is constitutive in L40 (inducible in EGY strains); selection is for HIS prototrophy. Integrated lacZ reporter is considerably less sensitive than pSH18‐34 maintained in EGY strains.

^f Interaction Trap reagents represent the work of many contributors. The original basic reagents were developed in the Brent laboratory (Gyuris et al., ). Strains for specialized applications have been developed by the following individuals: E. Golemis, Fox Chase Cancer Center, Philadelphia, Pa. (EGY48, EGY191); A.B. Vojtek and S.M. Hollenberg, Fred Hutchinson Cancer Research Center, Seattle, Wash. (L40).

^g Strains commercially available from OriGene.

Alternate Protocol 1: Confirmation of Fusion Protein Synthesis by Repression Assay

pBait ( protocol 1 )
pJK101 (Table 4.4.1 )
100‐mm complete minimal (CM) medium dropout plates (Treco and Lundblad, ) with 2% (w/v) glucose (Glu): Glu/CM −Ura

Basic Protocol 2: Performing an Interactor Hunt

Materials

Transformed yeast strains (see protocol 1 ), EGY48 containing:
- pSH18‐34 (lacZ reporter plasmid) and pBait (bait strain)
- pSH18‐34 and pRFHM‐1 (negative control)
- pSH18‐34 and any nonspecific bait (nonspecific control)
Complete minimal (CM) dropout liquid medium (Treco and Lundblad, ) with 2% (w/v) glucose (Glu) or 2% (w/v) galactose (Gal)/1% (w/v) raffinose (Raff):
- Glu/CM –Ura –His
- Gal/Raff/CM –Ura –His −Trp
- Gal/Raff/CM –Ura –His −Trp −Leu
H₂ O, sterile
TE buffer (pH 7.5; appendix 2A ), with and without 0.1 M lithium acetate
Library DNA in pJG4‐5 (Table 4.4.3 and Fig. )
High‐quality sheared salmon sperm DNA (see protocol 5 )
40% (w/v) polyethylene glycol 4000 (PEG 4000; filter sterilized) in 0.1 M lithium acetate/TE buffer (pH 7.5)
Dimethyl sulfoxide (DMSO)
Complete medium (CM) dropout plates (Treco and Lundblad, ; sizes indicated) with 2% (w/v) glucose (Glu) or 2% (w/v) galactose (Gal)/1% (w/v) raffinose (Raff), plus 20 µg/ml Xgal, as indicated:
- Glu/CM –Ura –His −Trp, 24 × 24‐cm (Nunc) and 100‐mm
- Gal/Raff/CM –Ura –His −Trp, 100‐mm
- Gal/Raff/CM –Ura –His −Trp −Leu, 100‐mm
- Glu/Xgal/CM –Ura –His −Trp, 100‐mm
- Gal/Raff/Xgal/CM –Ura –His −Trp, 100‐mm
- Glu/CM –Ura –His −Trp −Leu, 100‐mm
- Glu/CM –Ura –His, 100‐mm
Glycerol solution (see recipe )
Lysis solution (see recipe )
0.7% low‐melting agarose gel (see appendix 1N )
Hae III and appropriate enzyme buffer
10 µM forward primer (FP1): 5′‐CGT AGT GGA GAT GCC TCC‐3′
10 µM reverse primer (FP2): 5′‐CTG GCA AGG TAG ACA AGC CG‐3′
E. coli DH5α or other strain suitable for preparation of DNA for sequencing
Restriction enzymes: Eco R1 and Xho I
pJG4‐5 library vector (Fig. )

30°C incubator, with and without shaking
50‐ml conical tubes, sterile
1.5‐ml microcentrifuge tubes, sterile
42°C heating block
Glass microscope slides, sterile
Toothpicks or bacterial inoculating loop (unit 1.1 ), sterile
96‐well microtiter plate
Sealing tape, e.g., wide transparent tape
150‐ to 212‐µm glass beads, acid‐washed (soak 1 hr in concentrated nitric acid, rinse thoroughly with H₂ O, then oven dry)
Vortexer with plate adapters

Additional reagents and equipment for performing PCR (Kramer and Coen, ), agarose gel electrophoresis ( appendix 1N ), restriction endonuclease digestion ( appendix 1M ), bacterial transformation by electroporation (Seidman et al., ; optional), plasmid miniprep ( appendix 1J ; optional), and gap repair in yeast (Lundblad and Zhou, )

Alternate Protocol 2: Performing a Hunt by Interaction Mating

Yeast strains: RFY206 (MATa ura3 trp1 his3 leu2 ; Finley and Brent, )
YPD liquid medium and 100‐mm plates (Treco and Lundblad, )
Glu/CM −Trp dropout plates (Treco and Lundblad, ) with 2% glucose
pJG4‐5 library vector (Fig. )

Support Protocol 1: Preparation of Sheared Salmon Sperm Carrier DNA

Materials

High‐quality salmon sperm DNA (e.g., sodium salt from salmon testes, Sigma or Boehringer Mannheim), desiccated
TE buffer, pH 7.5 ( appendix 2A ), sterile
TE‐saturated buffered phenol ( appendix 2A )
1:1 (v/v) buffered phenol/chloroform
Chloroform
3 M sodium acetate, pH 5.2 ( appendix 2A )
100% and 70% (v/v) ethanol, ice cold

Magnetic stirring apparatus and stir‐bar, 4°C
Sonicator with probe
50‐ml conical centrifuge tube
High‐speed centrifuge and appropriate tube
100°C and ice‐water baths

Support Protocol 2: Yeast Colony Hybridization

Materials

Glu/CM −Trp plates: CM dropout plates −Trp (Treco and Lundblad, ) with 2% glucose
Master dropout plate of yeast positive for Gal dependence (see protocol 3 , step 19)
1 M sorbitol/20 mM EDTA/50 mM DTT (prepare fresh)
1 M sorbitol/20 mM EDTA
0.5 M NaOH
0.5 M Tris⋅Cl (pH 7.5)/6× SSC ( appendix 2A )
2× SSC ( appendix 2A )
100,000 U/ml β‐glucuronidase (type HP‐2 crude solution from Helix pomatia ; Sigma)

82‐mm circular nylon membrane, sterile
Whatman 3 MM paper
80°C vacuum oven or UV cross‐linker

Additional reagents and equipment for bacterial filter hybridization (Duby et al., ; Strauss, )

Support Protocol 3: Microplate Plasmid Rescue

Materials

2× Glu/CM −Trp liquid medium: 2× CM −Trp liquid medium (Treco and Lundblad, ) with 4% glucose
Master plate of Gal‐dependent yeast colonies (see protocol 3 , step 18)
Rescue buffer: 50 mM Tris⋅Cl (pH 7.5)/10 mM EDTA/0.3% (v/v) 2‐mercaptoethanol (prepare fresh)
Lysis solution: 2 to 5 mg/ml Zymolyase 100T/rescue buffer or 100,000 U/ml β‐glucuronidase (type HP‐2 crude solution from Helix pomatia ; Sigma) diluted 1:50 in rescue buffer
10% (w/v) SDS
7.5 M ammonium acetate ( appendix 2A )
Isopropanol
70% (v/v) ethanol
TE buffer, pH 8.0 ( appendix 2A )

24‐well microtiter plates
Centrifuge with rotor adapted for microtiter plates, refrigerated
Repeating micropipettor
37°C rotary shaker

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Figures

Figure 4.4.1 The interaction trap. (A ) An EGY48 yeast cell containing two LexA operator–responsive reporters, one a chromosomally integrated copy of the LEU2 gene (required for growth on−Leu medium), the second a plasmid bearing the lacZ reporter gene (causing yeast to turn blue on medium containing Xgal). The cell also contains a constitutively expressed chimeric protein, consisting of the DNA‐binding domain of LexA fused to the probe or bait protein, shown as being unable to activate either of the two reporters. (B ) and (C ) The resulting bait strain has been additionally transformed with an activation domain (act)–fused cDNA library in pJG4‐5, and the library has been induced. In (B), the encoded protein does not interact specifically with the bait protein and the two reporters are not activated. In (C), a positive interaction is shown in which the library‐encoded protein interacts with bait protein, resulting in activation of the two reporters (arrow), thus causing growth on medium lacking Leu and blue color on medium containing Xgal. Symbols: black rectangle, LexA operator sequence; open circle, LexA protein; open pentagon, bait protein; open rectangle, noninteracting library protein; shaded box, activator protein (acid blob in Fig. ); open chevron, interacting library protein.

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Figure 4.4.2 Flow chart for performing an interaction trap.

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Figure 4.4.3 LexA fusion plasmid pEG202. The strong constitutive ADH promoter is used to express bait proteins as fusions to the DNA‐binding protein LexA. Restriction sites shown in this map are based on pEG202 sequence data and include selected sites suitable for diagnostic restriction endonuclease digests. A number of restriction sites are available for insertion of coding sequences to produce protein fusions with LexA; the polylinker sequence and reading frame relative to LexA are shown below the map with unique sites marked in bold type. The sequence 5′‐CGT CAG CAG AGC TTC ACC ATT G‐3′ can be used to design a primer to confirm correct reading frame for LexA fusions. Plasmids contain the HIS3 selectable marker and the 2‐µm origin of replication to allow propagation in yeast, and the Ap^r antibiotic resistance gene and the pBR origin of replication to allow propagation in E. coli . In the plasmids pMW101 and pMW103, the ampicillin resistance gene (Ap^r ) has been replaced with the chloramphenicol resistance gene (Cm^r ) and the kanamycin resistance gene (Km^r ), respectively (see Table for details).

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Figure 4.4.4 lacZ reporter plasmid. pRB1840, pJK103, and pSH18‐34 are all derivatives of LR1Δ1 (West et al., ) containing eight, two, or one operator for LexA ( LexA _op ) binding inserted into the unique Xho I site located in the minimal GAL1 promoter ( GAL1 _pro ; 0.28 on map). The plasmid contains the URA3 selectable marker, the 2‐µm origin to allow propagation in yeast, the ampicillin resistance gene (Ap^r ), and the pBR322 origin (ori) to allow propagation in E. coli . Numbers indicate relative map positions. In the recently developed derivatives, the ampicillin resistance gene has been replaced with the chloramphenicol or kanamycin resistance genes (see Table for details).

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Figure 4.4.5 Repression assay for DNA binding. (A ) The plasmid pJK101 contains the upstream activating sequence (UAS) from the GAL1 gene followed by LexA operators (ops) upstream of the lacZ coding sequence. Thus, yeast containing pJK101 will have significant β‐galactosidase activity when grown on medium in which galactose is the sole carbon source because of binding of endogenous yeast GAL4 to the GAL _UAS . (B ) LexA‐fused proteins (P1‐LexA) that are made, enter the nucleus, and bind the LexA ops will block activation from the GAL _UAS , repressing β‐galactosidase activity (+) 3‐ to 5‐fold. On glucose/Xgal medium, yeast containing pJK101 should be white because GAL _UAS transcription is repressed.

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Figure 4.4.6 Library plasmid pJG4‐5. Library plasmids express cDNAs or other coding sequences inserted into unique Eco RI and Xho I sites as a translational fusion to a cassette consisting of the SV40 nuclear localization sequence (NLS; PPKKKRKVA), the acid blob B42 domain (Ruden et al, ), and the hemagglutinin (HA) epitope tag (YPYDVPDYA). Expression of cassette sequences is under the control of the GAL1 galactose‐inducible promoter. This map is based on the sequence data available for pJG4‐5, and includes selected sites suitable for diagnostic restriction digests (shown in bold). The sequence 5′‐CTG AGT GGA GAT GCC TCC‐3′ can be used as a primer to identify inserts or to confirm correct reading frame. The pJG4‐5 plasmid contains the TRP1 selectable marker and the 2‐µm origin to allow propagation in yeast, and the Ap^r antibiotic resistance gene and the pUC origin to allow propagation in E. coli . In the pJG4‐5 derivative plasmids pMW104 and pMW102, the ampicillin resistance gene has been replaced with the chloramphenicol resistance gene and kanamycin resistance gene, respectively (see Table for details). Currently existing libraries are all made in the pJG4‐5 plasmid (Gyuris et al., ) shown on this figure. Unique sites are marked in bold type.

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Literature Cited

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Key Reference
	Gyuris et al., 1993. See above.
	Initial description of interaction trap system.
Internet Resources
	http://cmmg.biosci.wayne.edu/rfinley/lab.html
	Source of two‐hybrid information, protocols, and links.
	http://www.origene.com
	Commercial source for basic plasmids, strains, and libraries for interaction trap experiments.
	brent@molsci.org
	Contacts for sources of interaction trap plasmids for specialized interactions.
	EA_Golemis@fccc.edu
	Database for false positive proteins detected in interaction trap experiments; analysis of two‐hybrid usage.
	http://www.fccc.edu:80/research/labs/golemis/InteractionTrapInWork.html

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Interaction Trap/Two‐Hybrid System to Identify Interacting Proteins

Abstract

Table of Contents

Materials

Basic Protocol 1: Producing and Characterizing a Bait Strain

Alternate Protocol 1: Confirmation of Fusion Protein Synthesis by Repression Assay

Basic Protocol 2: Performing an Interactor Hunt

Alternate Protocol 2: Performing a Hunt by Interaction Mating

Support Protocol 1: Preparation of Sheared Salmon Sperm Carrier DNA

Support Protocol 2: Yeast Colony Hybridization

Support Protocol 3: Microplate Plasmid Rescue

Figures

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Literature Cited