Interaction Trap/Two‐Hybrid System to Identify Loss‐of‐Interaction Mutant Proteins
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- Abstract
- Table of Contents
- Materials
- Figures
- 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.
Table of Contents
- Basic Protocol 1: Identifying Loss‐of‐Interaction Mutant Proteins
- Alternate Protocol 1: Create Prey Minilibrary by Conventional Cloning
- Commentary
- Literature Cited
- Figures
Materials
Basic Protocol 1: Identifying Loss‐of‐Interaction Mutant Proteins
Materials
Alternate Protocol 1: Create Prey Minilibrary by Conventional Cloning
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Figures
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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. View Image
Videos
Literature Cited
Literature Cited | |
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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. |