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

1 Fox Chase Cancer Center, Philadelphia, Pennsylvania, 2 Wayne State University School of Medicine, Detroit, Michigan, 3 Mitotix, Inc., Cambridge, Massachusetts, 4 The Molecular Sciences Institute, Berkeley, California
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 4.4
DOI:  10.1002/0471142301.ns0404s55
Online Posting Date:  April, 2011
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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. 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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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
  • 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
  • H 2O, 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 aa , bb   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 Apr Contains an ADH promoter that expresses LexA followed by polylinker
    pJK202 HIS3 Apr Like pEG202, but incorporates nuclear localization sequences between LexA and polylinker; used to enhance translocation of bait to nucleus
    pNLexA d HIS3 Apr 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 Apr 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 Apr An 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
    pRFHM1 d (control) HIS3 Apr 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 Apr ADH promoter expresses LexA fused to GAL4 activation domain; used as a positive control for transcriptional activation
    pMW101 e HIS3 Cmr Same as pEG202, but with altered antibiotic resistance markers; basic plasmid used for cloning bait
    pMW103 e HIS3 Kmr Same as pEG202, but with altered antibiotic resistance markers; basic plasmid used for cloning bait
    pHybLex/Zeo Zeor Zeor 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 Apr 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 Apr An 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
    pYESTrp TRP1 Apr 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 Kmr Same as pJG4‐5, but with altered antibiotic resistance markers; no libraries yet available
    pMW104 e TRP1 Cmr Same as pJG4‐5, but with altered antibiotic resistance markers; no libraries yet available
    LacZ reporter plasmids
    pSH18‐34 d URA3 Apr Contains eight LexA operators that direct transcription of the lacZ gene; one of the most sensitive indicator plasmids for transcriptional activation
    pJK103 d URA3 Apr Contains two LexA operators that direct transcription of the lacZ gene; an intermediate reporter plasmid for transcriptional activation
    pRB1840 d URA3 Apr Contains one LexA operator that directs transcription of the lacZ gene; one of the most stringent reporters for transcriptional activation
    pMW112 e URA3 Kmr Same as pSH18‐34, but with altered antibiotic resistance marker
    pMW109 e URA3 Kmr Same as pJK103, but with altered antibiotic resistance marker
    pMW111 e URA3 Kmr Same as pRB1840, but with altered antibiotic resistance marker
    pMW107 e URA3 Cmr Same as pSH18‐34, but with altered antibiotic resistance marker
    pMW108 e URA3 Cmr Same as pJK103, but with altered antibiotic resistance marker
    pMW110 e URA3 Cmr Same as pRB1840, but with altered antibiotic resistance marker
    pJK101 d (control) URA3 Apr 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 MATα 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 MATαtrp1, his3, ura3, lexAops‐LEU2 2 EGY191 provides a more stringent selection than EGY48, producing lower background with baits with instrinsic ability to activate transcription
    L40b MATα trpl, leu2, ade2, 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.

     aAll plasmids contain a 2µm 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., ). 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).
     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 4.4.2   Materials   Interaction Trap Plasmids aa , bb   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 Apr Contains an ADH promoter that expresses LexA followed by polylinker
    pJK202 HIS3 Apr Like pEG202, but incorporates nuclear localization sequences between LexA and polylinker; used to enhance translocation of bait to nucleus
    pNLexA d HIS3 Apr 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 Apr 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 Apr An 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
    pRFHM1 d (control) HIS3 Apr 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 Apr ADH promoter expresses LexA fused to GAL4 activation domain; used as a positive control for transcriptional activation
    pMW101 e HIS3 Cmr Same as pEG202, but with altered antibiotic resistance markers; basic plasmid used for cloning bait
    pMW103 e HIS3 Kmr Same as pEG202, but with altered antibiotic resistance markers; basic plasmid used for cloning bait
    pHybLex/Zeo Zeor Zeor 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 Apr 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 Apr An 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
    pYESTrp TRP1 Apr 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 Kmr Same as pJG4‐5, but with altered antibiotic resistance markers; no libraries yet available
    pMW104 e TRP1 Cmr Same as pJG4‐5, but with altered antibiotic resistance markers; no libraries yet available
    LacZ reporter plasmids
    pSH18‐34 d URA3 Apr Contains eight LexA operators that direct transcription of the lacZ gene; one of the most sensitive indicator plasmids for transcriptional activation
    pJK103 d URA3 Apr Contains two LexA operators that direct transcription of the lacZ gene; an intermediate reporter plasmid for transcriptional activation
    pRB1840 d URA3 Apr Contains one LexA operator that directs transcription of the lacZ gene; one of the most stringent reporters for transcriptional activation
    pMW112 e URA3 Kmr Same as pSH18‐34, but with altered antibiotic resistance marker
    pMW109 e URA3 Kmr Same as pJK103, but with altered antibiotic resistance marker
    pMW111 e URA3 Kmr Same as pRB1840, but with altered antibiotic resistance marker
    pMW107 e URA3 Cmr Same as pSH18‐34, but with altered antibiotic resistance marker
    pMW108 e URA3 Cmr Same as pJK103, but with altered antibiotic resistance marker
    pMW110 e URA3 Cmr Same as pRB1840, but with altered antibiotic resistance marker
    pJK101 d (control) URA3 Apr 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 MATα 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 MATαtrp1, his3, ura3, lexAops‐LEU2 2 EGY191 provides a more stringent selection than EGY48, producing lower background with baits with instrinsic ability to activate transcription
    L40b MATα trpl, leu2, ade2, 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.

     fInteraction 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).
     gStrains 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
  • H 2O, 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)
  • 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 DH5α or other strain suitable for preparation of DNA for sequencing
  • Restriction enzymes: EcoR1 and XhoI
  • 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 H 2O, 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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Literature Cited

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   Watson, M.A., Buckholz, R., and Weiner, M.P. 1996. Vectors encoding alternative antibiotic resistance for use in the yeast two‐hybrid system. BioTechniques 21:255‐259.
   West, R.W.J., Yocum, R.R., and Ptashne, M. 1984. Saccharomyces cerevisiae GAL1‐GAL10 divergent promoter region: Location and function of the upstream activator sequence UASG. Mol. Cell Biol. 4:2467‐2478.
   Yang, M., Wu, Z., and Fields, S. 1995. Protein‐peptide interactions analyzed with the yeast two‐hybrid system. Nucleic Acids Res. 23:1152‐1156.
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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