High‐Throughput Screening for Protein‐Protein Interactions Using Yeast Two‐Hybrid Arrays

Gerard Cagney1, Peter Uetz1

1 University of Washington, Seattle
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 19.6
DOI:  10.1002/0471140864.ps1906s24
Online Posting Date:  August, 2001
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Abstract

Arrays are used for parallel assays of large numbers of proteins in a single experiment. They provide an alternative to libraries for protein screening purposes. This unit describes a protein array composed of living yeast (Saccharomyces cerevisiae) cells that can be used in a functional screen for protein interactions, the two‚Äźhybrid assay. The protocols described here use the yeast proteome as an example, but they can be applied to any other genome or subset thereof.

     
 
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Table of Contents

  • Basic Protocol 1: Construction of a Proteome‐Scale Protein Array for Yeast
  • Basic Protocol 2: Manual Screening for Protein Interactions Using a Yeast Protein Array
  • Alternate Protocol 1: Robotic Screening for Protein Interactions Using a Yeast Protein Array
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Construction of a Proteome‐Scale Protein Array for Yeast

  Materials
  • DNAs encoding proteins of interest (e.g., genomic DNA, cDNA library)
  • Appropriate primers for amplification of the protein‐coding sequences
  • Two‐hybrid plasmids: pOAD and pOBD2 (Hudson et al., ; Cagney et al., ; also see )
  • NcoI and PvuII or other appropriate restriction enzymes, with buffers
  • recipeLiquid YEPD medium (see recipe)
  • S. cerevisiae host strains:
  •  PJ69‐4A (MATatrp1‐901 leu2‐3,112 ura3‐52 his3‐200 gal4Δ gal80ΔLYS2::GAL1‐HIS3 GAL2‐ADE2 met2::GAL7‐lacZ; James et al., )
  •  PJ69‐4α (MATαtrp1‐901 leu2‐3,112 ura3‐52 his3‐200 gal4Δ gal80ΔLYS2::GAL1‐HIS3 GAL2‐ADE2met2::GAL7‐lacZ; Uetz et al., )
  • 0.1 M lithium acetate
  • 7.75 mg/ml salmon sperm DNA (Sigma), autoclaved (15 min at 121°C) and stored at −20°C
  • recipe96PEG solution (see recipe)
  • Dimethyl sulfoxide (DMSO)
  • 35‐mm plates containing solid −Leu (for pOAD) and −Trp (for pOBD2) recipedropout medium (see recipe)
  • Liquid −Leu recipedropout medium (see recipe)
  • Single‐well microtiter plates (e.g., OmniTray, Nalge Nunc International) containing solid −Leu recipedropout medium (see recipe)
  • 40% (v/v) glycerol
  • 250‐ml conical flasks
  • 30°C shaking incubator
  • 8‐channel pipettor (to deliver 200 µl) and sterile pipetting troughs
  • 96‐ and 384‐well microtiter plates (e.g., Nalge Nunc International)
  • Plastic adhesive tape for sealing plates
  • 42°C incubator
  • Centrifuge with insert for 96‐well plates
  • Toothpicks, sterile
  • 384‐Pin Replicator (Nalge Nunc International)
  • 2‐ml cryotubes (e.g., Nalge Nunc International)
  • Additional reagents and equipment for PCR amplification ( appendix 4J), agarose gel electrophoresis ( appendix 4F), digestion of DNA with restriction endonucleases ( appendix 4I), and gel purification ( appendix 4F) and quantification ( appendix 4K) of DNA

Basic Protocol 2: Manual Screening for Protein Interactions Using a Yeast Protein Array

  Materials
  • 20% (v/v) bleach (∼1% sodium hypochlorite)
  • 95% (v/v) ethanol
  • Yeast protein array (see protocol 1)
  • Single‐well microtiter plates (e.g., OmniTray; Nalge Nunc International) containing solid recipeYEPD +Ade medium (see recipe), recipeYEPD medium (see recipe), −Leu −Trp recipedropout medium (see recipe), and −His −Leu −Trp +3AT recipedropout medium (see recipe)
  • recipeLiquid YEPD medium (see recipe)
  • DBD fusion–expressing yeast strain (see protocol 1)
  • 384‐Pin Replicator (Nalge Nunc International)
  • 30°C incubator
  • 250‐ml conical flask
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Figures

Videos

Literature Cited

Literature Cited
   Cagney, G., Uetz, P., and Fields, S. 2000. High‐throughput screening for protein‐protein interactions using two‐hybrid assay. Methods Enzymol. 328:3‐14.
   Fields, S. and Song, O. 1989. A novel genetic system to detect protein‐protein interactions. Nature 340:245‐246.
   Flajolet, M., Rotondo, G., Daviet, L., Bergametti, F., Inchauspe, G., Tiollais, P., Transy, C., and Legrain, P. 2000. A genomic approach of the hepatitis C virus generates a protein interaction map. Gene 242:369‐379.
   Fromont‐Racine, M., Rain, J.C., and Legrain, P. 1997. Toward a functional analysis of the yeast genome through exhaustive two‐hybrid screens. Nature Genet. 16:277‐282.
   Hudson, J.R., Dawson, E.P., Rushing, K.L., Jackson, C.H., Lockshon, D., Conover, D., Lanciault, C., Harris, J.R., Simmons, S.J., Rothstein, R., and Fields, S. 1997. The complete set of predicted genes from Saccharomyces cerevisiae in a readily usable form. Genome Res. 7:1169‐1173.
   James, P., Halladay, J., and Craig, E.A. 1996. Genomic libraries and a host strain designed for highly efficient two‐hybrid selection in yeast. Genetics. 144:1425‐1436.
   Kraemer, B., Zhang, B., SenGupta, D., Fields, S., and Wickens, M. 2000. Using the yeast three‐hybrid system to detect and analyze RNA‐protein interactions. Methods Enzymol. 328:297‐321.
   Orr‐Weaver, T.L., Szostak, J.W., and Rothstein, R.J. 1981. Yeast transformation: A model system for the study of recombination. Proc. Natl. Acad. Sci. U.S.A. 78:6354‐6358.
   Rain, J.C., Selig, L., De Reuse, H., Battaglia, V., Reverdy, C., Simon, S., Lenzen, G., Petel, F., Wojcik, J., Schachter, V., Chemama, Y., Labigne, A., and Legrain, P. 2001. The protein‐protein interaction map of Helicobacter pylori. Nature 409:211‐215.
   Uetz, P., Hughes, R.E., and Fields, S. 1998. The two‐hybrid system: Finding likely partners for lonely proteins. Focus (Life Technologies) 20:62‐65.
   Uetz, P., Giot, L., Cagney, G., Mansfield, T., Judson, R., Knight, J., Lockshon, D., Narayan, V., Srinivasan, M., Pochart, P., Qureshi‐Emili, A., Li, Y., Godwin, B., Conover, D., Kalbfleisch, T., Vijayadamodar, G., Yang, M., Johnston, M., Fields, S., and Rothberg, J. 2000. A comprehensive analysis of protein‐protein interactions in Saccharomyces cerevisiae. Nature 403:623‐627.
   Vidal, M. and Legrain, P. 1999. Yeast forward and reverse “n”‐hybrid systems. Nucl. Acids Res. 27:919‐929.
Key Reference
   Uetz et al., 2000. See above.
  This paper, the first to describe two‐hybrid arrays, provides the results of 192 screens and compares them to screens that used a library of preys.
Internet Resources
   http://depts.washington.edu/sfields/
  The Fields laboratory Web site contains more details on primer design, vectors, and array screening as well as results from several hundred screens.
   http://www.lab‐robotics.org/
  The Laboratory Robotics Interest Group Web site provides a guide to robotics manufacturers and product information.
   http://www.proteome.com
  Proteome's Web site includes a Yeast Protein Database.
   http://mips.gsf.de/proj/yeast/
  The Munich Information Center for Protein Sequences Web site provides yeast protein databases and information on other genomes and proteomes.
   http://www.fccc.edu/research/labs/golemis/InteractionTrapInWork.html
  The Golemis laboratory Web site offers general information on two‐hybrid screens and false positives.
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