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Interaction Trap/Two‐Hybrid System to Identify Interacting Proteins

Erica A. Golemis1,  Ilya Serebriiskii1,  Russell L. Finley2,  Mikhail G. Kolonin (hunt by interaction mating)2,  Jeno Gyuris3,  Roger Brent4

1Fox Chase Cancer Center, Philadelphia, Pennsylvania
2Wayne State University School of Medicine, Detroit, Michigan
3Mitotix, Inc., Cambridge, Massachusetts
4The Molecular Sciences Institute, Berkeley, California




Publication Name: 
Current Protocols in Protein Science
Unit Number: 
Unit 19.2
DOI: 
10.1002/0471140864.ps1902s57
Online Posting Date: 
August, 2009
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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. Protein Sci. 57:19.2.1-19.2.35. © 2009 by John Wiley & Sons, Inc.

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

     
 
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Table of Contents

  • 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
  • Bibliography
  • 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 19.2.1): e.g., pEG202 (Fig. 19.2.3), pSH18-34 (Fig. 19.2.4), pSH17-4, pRFHM1
  • Yeast strain: e.g., EGY48 (ura3 trp1 his3 3LexA-operator-LEU2; see Table 19.2.2)
  • 100-mm complete minimal (CM) medium dropout plates (unit 13.1) 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 (appendix 4L)
  • CM dropout liquid medium (appendix 4L) with 2% (w/v/) glucose: Glu/CM –Ura –His
  • 2× Laemmli sample buffer (see recipe)
  • Antibody to LexA or fusion domain
  • H2O, sterile
  • 30°C incubator
  • 100°C water bath
  • Additional reagents and equipment for subcloning (Struhl, 1991), lithium acetate transformation of yeast (appendix 4L), filter lift or liquid assay for -galactosidase (appendix 4L), SDS-PAGE (unit 10.1), and immunoblotting (unit 10.10)
     
    Table 19.2.1 Interaction Trap Plasmidsa,b
    Selection 
    Plasmid name/sourceIn yeastIn E. coliComment/description
    LexA fusion plasmids
    pEG202c,dHIS3AprContains an ADH promoter that expresses LexA followed by polylinker
    pJK202HIS3AprLike pEG202, but incorporates nuclear localization sequences between LexA and polylinker; used to enhance translocation of bait to nucleus
    pNLexAdHIS3AprContains an ADH promoter that expresses polylinker followed by LexA for use with baits where their amino-terminal residues must remain unblocked
    pGildadHIS3AprContains a GAL1 promoter that expresses same LexA and polylinker cassette as pEG202 for use with baits where their continuous presence is toxic to yeast
    pEE202IHIS3AprAn integrating form of pEG202 that can be targeted into HIS3 following digestion with KpnI; for use where physiological screen requires lower levels of bait to be expressed
    pRFHM1d (control)HIS3AprContains 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
    pSH17-4d (control)HIS3AprADH promoter expresses LexA fused to GAL4 activation domain; used as a positive control for transcriptional activation
    pMW101eHIS3CmrSame as pEG202, but with altered antibiotic resistance markers; basic plasmid used for cloning bait
    pMW103eHIS3KmrSame as pEG202, but with altered antibiotic resistance markers; basic plasmid used for cloning bait
    pHybLex/ZeoZeorZeorBait 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
    pJG4-5c,dTRP1AprContains a GAL1 promoter that expresses nuclear localization domain, transcriptional activation domain, HA epitope tag, cloning sites; used to express cDNA libraries
    pJG4-5ITRP1AprAn integrating form of pJG4-5 that can be targeted into TRP1 by digestion with Bsu36I (New England Biolabs); to be used with pEE202I to study interactions that occur physiologically at low protein concentrations
    pYESTrpbTRP1AprContains 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)
    pMW102eTRP1KmrSame as pJG4-5, but with altered antibiotic resistance markers; no libraries yet available
    pMW104eTRP1CmrSame as pJG4-5, but with altered antibiotic resistance markers; no libraries yet available
    LacZ reporter plasmids
    pSH18-34dURA3AprContains eight LexA operators that direct transcription of the lacZ gene; one of the most sensitive indicator plasmids for transcriptional activation
    pJK103dURA3AprContains two LexA operators that direct transcription of the lacZ gene; an intermediate reporter plasmid for transcriptional activation
    pRB1840dURA3AprContains one LexA operator that directs transcription of the lacZ gene; one of the most stringent reporters for transcriptional activation
    pMW112eURA3KmrSame as pSH18-34, but with altered antibiotic resistance marker
    pMW109eURA3KmrSame as pJK103, but with altered antibiotic resistance marker
    pMW111eURA3KmrSame as pRB1840, but with altered antibiotic resistance marker
    pMW107eURA3CmrSame as pSH18-34, but with altered antibiotic resistance marker
    pMW108eURA3CmrSame as pJK103, but with altered antibiotic resistance marker
    pMW110eURA3CmrSame as pRB1840, but with altered antibiotic resistance marker
    pJK101d (control)URA3AprContains 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
     aAll plasmids contain a 2 µm origin for maintenance in yeast, as well as a bacterial origin of replication, except where noted (pEE202I, pJG4-5I).
     bInteraction Trap reagents represent the work of many contributors: the original basic reagents were developed in the Brent laboratory (Gyuris et al., 1993). Plasmids with altered antibiotic resistance markers (all pMW plasmids) were constructed at Glaxo in Research Triangle Park, N.C. (Watson et al., 1996). 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).
     cSequence data are available for pEG202 (pLexA), accession number U89960, and pJG4-5, accession number U89961.
     dPlasmids and strains available from OriGene.
     eIn pMW plasmids the ampicillin resistance gene (Apr) is replaced with the chloramphenicol resistance gene (Cmr) and the kanamycin resistance gene (Kmr) from pBC SK(+) and pBK-CMV (Stratagene), respectively. The choice between Kmr and Cmr or Apr plasmids is a matter of personal taste; use of basic Apr 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. Apr 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 19.2.2 Interaction Trap Yeast Selection Strainsa
    StrainRelevant genotypeNumber of operatorsComments/description
    EGY48bMAT trp1, his3, ura3, lexAops-LEU26lexA operators direct transcription from the LEU2 gene; EGY48 is a basic strain used to select for interacting clones from a cDNA library
    EGY191MAT trp1, his3, ura3, lexAops-LEU22EGY191 provides a more stringent selection than EGY48, producing lower background with baits with instrinsic ability to activate transcription
    L40bMAT trpl, leu2, ade2, GAL4, lexAops-HIS34, lexAops-lacZ8Expression 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.
     aInteraction Trap reagents represent the work of many contributors. The original basic reagents were developed in the Brent laboratory (Gyuris et al., 1993). 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).
     bStrains commercially available from OriGene.

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

 Additional Materials (also see Basic Protocol 1)
  • pBait (Basic Protocol 1)
  • pJK101 (Table 19.2.1)
  • 100-mm complete minimal (CM) medium dropout plates (appendix 4L) with 2% (w/v) glucose (Glu): Glu/CM –Ura

Basic Protocol 2: Performing an Interactor Hunt

 Materials
  • Transformed yeast strains (see Basic 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 (appendix 4L) 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
  • H2O, sterile
  • TE buffer (pH 7.5; appendix 2E), with and without 0.1 M lithium acetate
  • Library DNA in pJG4-5 (Table 19.2.3 and Fig. 19.2.6)
  • High-quality sheared salmon sperm DNA (see Support Protocol 1)
  • 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 (appendix 4L; 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 4F)
  • HaeIII 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 DH5 or other strain suitable for preparation of DNA for sequencing
  • Restriction enzymes: EcoR1 and XhoI
  • pJG4-5 library vector (Fig. 19.2.6)
  • 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, sterile
  • 96-well microtiter plate
  • Sealing tape, e.g., wide transparent tape
  • 150- to 212-µm glass beads, acid-washed
  • Vortexer with plate adapters
  • Additional reagents and equipment for performing PCR (appendix 4J), agarose gel electrophoresis (appendix 4F), restriction endonuclease digestion (appendix 4I), bacterial transformation by electroporation (unit 5.10; optional), and plasmid miniprep (appendix 4C; optional)

Alternate Protocol 2: Performing a Hunt by Interaction Mating

 Additional Materials (also see Basic Protocols 1 and 2)
  • Yeast strains: RFY206 (MATa ura3 trp1 his3 leu2; Finley and Brent, 1994)
  • YPD liquid medium and 100-mm plates (appendix 4L)
  • Glu/CM –Trp dropout plates (appendix 4L) with 2% glucose
  • pJG4-5 library vector (Fig. 19.2.6)

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 2E), sterile
  • TE-saturated buffered phenol (appendix 2E)
  • 1:1 (v/v) buffered phenol/chloroform
  • Chloroform
  • 3 M sodium acetate, pH 5.2 (appendix 2E)
  • 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 (appendix 4L) with 2% glucose
  • Master dropout plate of yeast positive for Gal dependence (see Basic Protocol 2, 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 2E)
  • 2× SSC (appendix 2E)
  • 100,000 U/ml -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 (Strauss, 1991; Duby et al., 1990)

Support Protocol 3: Microplate Plasmid Rescue

 Materials
  • 2× Glu/CM –Trp liquid medium: 2× CM –Trp liquid medium (appendix 4L) with 4% glucose
  • Master plate of Gal-dependent yeast colonies (see Basic Protocol 2, step )
  • 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 -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
  • Isopropanol
  • 70% (v/v) ethanol
  • TE buffer, pH 8.0 (appendix 2E)
  • 24-well microtiter plates
  • Centrifuge with rotor adapted for microtiter plates, refrigerated
  • Repeating micropipettor
  • 37°C rotary shaker
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Figures

  • Figure 19.2.1
    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. 19.2.6); open chevron, interacting library protein.

    View Image
  • Figure 19.2.2
    Flow chart for performing an interaction trap.

    View Image
  • Figure 19.2.3
    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 Apr 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 (Apr) has been replaced with the chloramphenicol resistance gene (Cmr) and the kanamycin resistance gene (Kmr), respectively (see Table 19.2.1 for details).

    View Image
  • Figure 19.2.4
    lacZ reporter plasmid. pRB1840, pJK103, and pSH18-34 are all derivatives of LR11 (West et al., 1984) containing eight, two, or one operator for LexA (LexAop) binding inserted into the unique XhoI site located in the minimal GAL1 promoter (GAL1pro; 0.28 on map). The plasmid contains the URA3 selectable marker, the 2-µm origin to allow propagation in yeast, the ampicillin resistance gene (Apr), 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 19.2.1 for details).

    View Image
  • Figure 19.2.5
    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 -galactosidase activity when grown on medium in which galactose is the sole carbon source because of binding of endogenous yeast GAL4 to the GALUAS. (B) LexA-fused proteins (P1-LexA) that are made, enter the nucleus, and bind the LexA ops will block activation from the GALUAS, repressing -galactosidase activity (+) 3- to 5-fold. On glucose/Xgal medium, yeast containing pJK101 should be white because GALUAS transcription is repressed.

    View Image
  • Figure 19.2.6
    Library plasmid pJG4-5. Library plasmids express cDNAs or other coding sequences inserted into unique EcoRI and XhoI 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., 1991), 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 Apr 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 19.2.1 for details). Currently existing libraries are all made in the pJG4-5 plasmid (Gyuris et al., 1993) shown on this figure. Unique sites are marked in bold type.

    View Image

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 Key Reference
    Gyuris et al., 1993. See above.

Initial description of interaction trap system.

 Internet Resources
    http://proteome.wayne.edu/finlabindex.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
    EA_Golemis@fccc.edu

Contacts for sources of interaction trap plasmids for specialized interactions.

    http://www.fccc.edu/research/labs/golemis/InteractionTrapInWork.html

Database for false positive proteins detected in interaction trap experiments; analysis of two-hybrid usage.

     
 
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