Phage‐Based Expression Cloning to Identify Interacting Proteins

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Abstract
Table of Contents
Materials
Figures
Literature Cited

Abstract

Phage?based expression cloning is a simple, rapid, and powerful technique to identify interacting proteins. A protein of interest is expressed as a recombinant fusion protein and labeled with ³² P at an engineered recognition site to facilitate detection. b?gal proteins that are fused in?frame to cDNA inserts in a phage?derived expression library are produced by the phage and adsorbed onto nitrocellulose filters. The filters are then screened with the radioactive protein probe to identify phage clones that express the interacting protein. This technique leads directly to the isolation of a cDNA encoding the interacting protein, bypassing the need for labor?intensive protein purification, microsequencing, or antibody production.

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Strategic Planning
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‐³² P]ATP (6000 mCi/mmol)
Purified glutathione‐S ‐transferase (GST)–bait protein fusion protein with a PKA recognition site (unit 16.7 ), 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 (see unit 1.1 appendix 3F , and Table 97.80.4711 ), 10 mM MgSO₄ , and 0.2% maltose
10 mM MgSO₄
10 mM IPTG (Table 97.80.4711 )
150‐ or 100‐mm LB plates (with antibiotic, if necessary; unit 1.1 )
0.7% top agarose (unit 1.1 ), 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 )
Suspension medium (SM ; unit 6.12 )
Chloroform

3‐ml disposable plastic columns or disposable syringe and glass wool
Scintillation counter and fluid
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 16.7 ), SDS‐PAGE (optional; unit 10.2 ), autoradiography ( appendix 3A ), titering and plating λ phage to generate plaques (unit 1.11 & unit 6.1 ), and purification of bacteriophage clones (unit 6.5 )

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Figures

Figure 20.3.1 Schematic representation of the interaction cloning technique used to identify proteins that associate with a protein of interest (bait protein), and the expected results of a successful screen. (A ) Expression of β‐galactosidase fusion proteins from in‐frame cDNA inserts of a λgt11 Arabidopsis library is induced with IPTG‐impregnated nitrocellulose membrane filters (indicated by an oval). Filters are probed with GST‐bait fusion protein labeled with ³² P (⋅) at the PKA recognition site located at the junction of the fusion. Interacting clones are detected by autoradiography. (B ) Representative autoradiogram of a tertiary screen of a positive plaque after the control experiment to determine whether the interaction is specific for the bait portion of the probe. The top half of the filter is probed with GST‐bait while the bottom half is probed with GST alone as a control.

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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.
	Lester, L.B., Coghlan, V.M., Nauert, B., and Scott, J.D. 1996. Cloning and characterization of a novel A‐kinase anchoring protein: AKAP220, association with testicular peroxisomes. J. Biol. Chem. 271:9460‐9465.
	Lowenstein, E.J., Daly, R.J., Batzer, A.G., Li, W., Margolis, B., Lammers, R., Ullrich, A., Skolnik, E.Y., Bar‐Sagi, D., and Schlessinger, J. 1992. The SH2 and SH3 domain‐containing protein GRB2 links receptor tyrosine kinases to ras signaling. Cell 70:431‐442.
	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.
	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 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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Phage‐Based Expression Cloning to Identify Interacting Proteins

Abstract

Table of Contents

Materials

Basic Protocol 1: Interaction Cloning

Figures

Videos

Literature Cited