Interaction Trap/Two‐Hybrid System to Identify Loss‐of‐Interaction Mutant Proteins

互联网2013-12-31

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Abstract
Table of Contents
Materials
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Literature Cited

Abstract

Protein?protein interactions play a critical role in biology. Disrupting a specific protein?protein interaction and studying the resulting phenotype can help elucidate the function of the interaction. A method to rapidly make and identify mutant proteins that no longer bind to a specific partner protein is described. The method takes advantage of the ease of the interaction trap/two?hybrid system and PCR mutagenesis. PCR fusion of the target protein to GFP is used to ensure the protein's open reading frame is not disrupted by mutagenesis. The resulting noninteracting mutant proteins usually result from a single missense mutation, which can easily be identified.

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Basic Protocol 1: Identifying Loss‐of‐Interaction Mutant Proteins
Alternate Protocol 1: Create Prey Minilibrary by Conventional Cloning
Commentary
Literature Cited
Figures

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Materials

Basic Protocol 1: Identifying Loss‐of‐Interaction Mutant Proteins

Materials

Genes for proteins of interest
Two‐hybrid interaction trap plasmids (Table 97.80.4711 ):
- Prey plasmid pJG4‐5
- Bait plasmid pEG202
- LacZ reporter plasmid pSH18‐34
Two‐hybrid interaction trap yeast strain EGY48 (unit 20.1 )
CM dropout plates (unit 13.1 ) substituted with 2% glucose (Glc), 2% galactose(Gal), and/or 1% raffinose (Raff) as indicated:
- Glc/CM −Ura
- Glc/CM −Ura −His
- Glc/CM −Ura −His, −Trp
- Glc/CM/Xgal −Ura −His −Trp
- Gal/Raff/CM/20 µg/ml Xgal in Z buffer −Ura, −His, −Trp: prepare as described for CM plates (unit 13.1 ) using a 1 mg/ml Xgal stock (20 µg/ml final) in Z buffer (unit 13.6 )
2.5 mM (each) dNTP stock solution (unit 15.1 )
2.5 U/µl high‐fidelity PCR‐compatible DNA polymerase (e.g., Pfu Turbo, Kod) and 10× buffer
1 U/µl Taq DNA polymerase (unit 3.5 ) and 10× Mg²⁺ ‐free reaction buffer
2.5 mM MgCl₂
50 mM MnCl₂
Plasmid carrying EGFP (e.g., pEGFP; BD Biosciences)
CM −Trp liquid medium (unit 13.1 )
TrpC E. coli strain KC8 (hsd R, leu B600, trp C9830, pyr F::Tn5, his B463, lac ΔX74, str A, gal U, gal K) (unit 20.1 ; constructed by K. Struhl and available from R. Brent), competent
M9 minimal plates (unit 1.1 ) containing 100 µg/ml amp, 40 µg/ml Leu, 40 µg/ml His, and 40 µg/ml Ura
Commercial miniprep kit (e.g., Qiagen; optional)
30°C incubator
Long‐wavelength UV lamp, or fluorescence microscope equipped with fluorescein or GFP filter set

Additional reagents and equipment for subcloning into plasmid vectors (unit 3.16 ), lithium acetate transformation (unit 13.7 ), PCR (unit 15.1 ), agarose gel electrophoresis (unit 20.5 ), restriction digestion (unit 3.1 ), replica plating using velvet filters (units 1.3 & 13.2 ), interaction trap (unit 20.1 ), purification of DNA by glass beads (unit 3.11 ), DNA minipreps (unit 2.1 ; optional), bacterial transformation (unit 1.8 ), sequencing DNA by dideoxy chain termination (unit 7.4 ), and site‐directed PCR mutagenesis (unit 8.5 )

Alternate Protocol 1: Create Prey Minilibrary by Conventional Cloning

T4 DNA ligase (unit 3.14 )
LB medium and plates containing amp (units 1.1 & 1.4 )

Additional reagents and equipment for gel purification of DNA fragments (unit 2.6 ), multiple endonuclease digestion (unit 3.2 ), DNA ligation (units 3.14 & 3.16 )

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Figures

Figure 20.8.1 Interaction trap/two‐hybrid system loss‐of‐interaction mutant proteins. Circled numbers indicate primers (see ). The asterisk ~undefined) on primer 2 indicates that the stop codon has been deleted from the ORF.

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Literature Cited

Literature Cited
	Cadwell, R.C. and Joyce, G.F. 1992. Randomization of genes by PCR mutagenesis. PCR Methods Appl. 2:28‐33.
	Gyuris, J., Golemis, E., Chertkov, H., and Brent, R. 1993. Cdi1, a human G1 and S phase protein phosphatase that associates with Cdk2. Cell 75:791‐803.
	Lin‐Goerke, J.L., Robbins, D.J., and Burczak, J.D. 1997. PCR‐based random mutagenesis using manganese and reduced dNTP concentration. Biotechniques 23:409‐412.
	Mendelsohn, A.R., Hamer, J.D., Wang, Z.B., and Brent, R. 2002. Cyclin D3 activates caspase 2, connecting cell proliferation with cell death. Proc. Natl. Acad. Sci. U.S.A. 99:6871‐6876.

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Interaction Trap/Two‐Hybrid System to Identify Loss‐of‐Interaction Mutant Proteins

Abstract

Table of Contents

Materials

Basic Protocol 1: Identifying Loss‐of‐Interaction Mutant Proteins

Alternate Protocol 1: Create Prey Minilibrary by Conventional Cloning

Figures

Videos

Literature Cited