Phage‐Based Expression Cloning to Identify Interacting Proteins

Julie M. Stone1

1 Massachusetts General Hospital, Boston
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 19.3
DOI:  10.1002/0471140864.ps1903s15
Online Posting Date:  May, 2001
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Abstract

Interaction cloning (also known as expression cloning) is a technique to identify and clone genes which encode proteins that interact with a protein of interest, or “bait” protein. The procedure presented in this unit involves a fusion protein consisting of bait protein and glutathione‐S‐transferase (GST) with a protein kinase A site at the junction between them (the protocol can, however, be adapted to use other PKA‐containing recombinant proteins). The labeled protein is subsequently used as a probe to screen a l bacteriophage‐derived cDNA expression library, which expresses b ‐galactosidase fusion proteins that contain in‐frame gene fusions. The phages lyse cells, form plaques, and release fusion proteins that are adsorbed onto nitrocellulose membrane filters. The filters are blocked with excess nonspecific protein to eliminate nonspecific binding and probed with the radiolabeled bait protein. This procedure leads directly to the isolation of genes encoding the interacting protein, bypassing the need for purification and microsequencing or for antibody production.

     
 
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Table of Contents

  • Basic Protocol 1: Interaction Cloning
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Interaction Cloning

  Materials
  • cAMP‐dependent protein kinase (PKA; e.g., 250‐U lots from Sigma)
  • 40 mM DTT, prepared fresh
  • recipe10× PKA buffer (see recipe)
  • 10 mCi/ml [γ‐32P]ATP (6000 mCi/mmol)
  • Purified glutathione‐S‐transferase (GST)–bait protein fusion protein with a PKA recognition site (unit 6.6), at ∼0.1 to 1 µg/µl concentration
  • recipeZ′‐KCl (see recipe), ice cold
  • Sephadex G‐50 equilibrated in recipeZ′‐KCl
  • E. coli Y1090r or other appropriate host strain
  • LB medium containing appropriate selective antibiotic ( appendix 4B), 10 mM MgSO 4, and 0.2% maltose
  • 10 mM MgSO 4
  • 10 mM IPTG (Table 97.80.4711)
  • 150‐ or 100‐mm LB plates (with antibiotic, if necessary; appendix 4B)
  • 0.7% top agarose ( appendix 4B), 47°C
  • recipeTris‐buffered saline with Triton X‐100 (TBS‐T; see recipe)
  • India ink
  • recipeHEPES blocking buffer (HBB; see recipe)
  • recipeBinding buffer (BB; see recipe)
  • recipeSuspension medium (SM; see recipe)
  • Chloroform
  • 3‐ml disposable plastic columns or disposable syringe and glass wool
  • Scintillation counter
  • Tabletop centrifuge or equivalent
  • Nitrocellulose membrane filters (137‐ and 82‐mm disks)
  • 22‐G needle
  • Additional reagents and equipment for preparation and purification of recombinant glutathione‐S‐transferase fusion protein (unit 6.6), SDS‐PAGE (optional; unit 10.1), autoradiography (unit 10.11), titering and plating λ phage to generate plaques (Lech and Brent, ; Quertermous, ), and purification of bacteriophage clones (Quertermous, )
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Figures

Videos

Literature Cited

Literature Cited
   Blanar, M.A. and Rutter, W.J. 1992. Interaction cloning: Identification of a helix‐loop‐helix zipper protein that interacts with c‐Fos. Science 256:1014‐1018.
   Carr, D.W. and Scott, J.D. 1992. Blotting and band‐shifting: Techniques for studying protein‐protein interactions. Trends Biochem.Sci. 17:246‐249.
   Chapline, C., Ramsay, K., Klauck, T., and Jaken, S. 1993. Interaction cloning of protein kinase C substrates. J. Biol. Chem. 268:6858‐6861.
   Hoeffler, J.B., Lustbader, J.W., and Chen, C.Y. 1991. Identification of multiple nuclear factors that interact with cyclic AMP response element‐binding protein and activation transcription factor‐2 by protein interactions. Mol. Endocrinol. 5:256‐266.
   Huynh, T.V., Young, R.A., and Davis, R.W. 1985. Constructing and screening cDNA libraries in λgt10 and λgt11. In DNA Cloning: A Practical Approach (D.M. Glover, ed.) pp. 49‐78. IRL Press, Oxford.
   Kaelin, W.G.J., Krek, W., Sellers, W.R., DeCaprio, J.A., Ajchenbaum, F., Fuchs, C.S., Chittenden, T., Li, Y., Farnham, P.J., Blanar, M.A., Livingston, D.M., and Flemington, E.K. 1992. Expression cloning of a cDNA encoding a retinoblastoma‐binding protein with E2F‐like properties. Cell 70:351‐364.
   Klickstein, L.B. and Neve, R. L. 1991. Ligation of linkers or adapters to double‐stranded cDNA. In Current Protocols in Molecular Biology (F.A. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp.5.6.1‐5.6.10. John Wiley & Sons, New York.
   Klickstein, L.B., Neve, R.L., Golemis, E.A., and Gyuris, J. 1995. Conversion of mRNA into double‐stranded cDNA. In Current Protocols in Molecular Biology. (F.A. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp.5.5.1‐5.5.14. John Wiley & Sons, New York.
   Lech, K. and Brent, R. 1988. Plating lambda phage to generate plaques. In Current Protocols in Molecular Biology (F.A. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp.1.11.1‐1.11.4 John Wiley & Sons, New York.
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   Margolis, B., Silvennoinen, O., Comoglio, F., Roonprapunt, C., Skolnik, E., Ullrich, A., and Schlessinger, J. 1992. High‐efficiency expression/cloning of epidermal growth factor–receptor‐binding proteins with src homology 2 domains. Proc. Natl. Acad. Sci. U.S.A 89:8894‐8898.
   Quertermous, T. 1987. Purification of bacteriophage clones. In Current Protocols in Molecular Biology (F.A. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp.6.5.1‐6.5.2. John Wiley & Sons, New York
   Quertermous, T. 1996. Plating and transferring bacteriophage libraries. In Current Protocols in Molecular Biology (F.A. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp.6.1.1‐6.1.4. John Wiley & Sons, New York.
   St. John, T. P. 1990. Immunoscreening of fusion proteins produced in lambda plaques. In Current Protocols in Molecular Biology (F.A. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp.6.7.1‐6.7.6. John Wiley & Sons, New York.
   Singh, H. 1991. Detection, purification, and characterization of cDNA clones encoding DNA‐binding proteins. In Current Protocols in Molecular Biology (F.A. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp.12.7.1‐12.7.10. John Wiley & Sons, New York.
   Skolnik, E.Y., Margolis, B., Mohammadi, M., Lowenstein, E., Fischer, R., Drepps, A., Ullrich, A., and Schlessinger, J. 1991. Cloning of PI3 kinase–associated p85 utilizing a novel method of expression/cloning of target proteins for receptor tyrosine kinases. Cell 65:83‐90.
   Stone, J.M., Collinge, M.A., Smith, R.D., Horn, M.A. and Walker, J.C. 1994. Interaction of a protein phosphatase with an Arabidopsis serine‐threonine receptor kinase. Science 266:793‐795.
   Vinson, C.R., LaMarco, K.L., Johnson, P.F., Landschulz, W.H., and McKnight, S.L. 1988. In situ detection of sequence‐specific DNA binding activity specified by a recombinant bacteriophage. Genes.& Dev. 2:801‐806
Key References
   Blanar and Rutter, 1992. See above.
  The basic protocol described in this unit is modified directly from the Blanar and Rutter protocol.
   Huynh et al., 1985. See above.
  Provides an excellent description of constructing and screening λgt11 cDNA expression libraries.
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