The chimeric proteins are made as translation gene fusionsin yeast. Two plasmids are made, one in which a chimeric protein is fused to the binding domain, the other where the protein is fused to the activating domain.These plasmids are then transformed into a suitable yeast strain and the individual chimeric proteins are produced in this cell.

Applications

You can use the system to: 

1. test for interactions between two proteins for which you have the genes. 
2. as a means of cloning a gene encoding a protein which interacts with a protein for which you have the gene. 

The gene you have is called the bait. The gene you catch with the bait is the prey. This is achieved by making a cDNA library in a special vector so that the yeast translates the gene you have cloned as a cDNA as a fusion with the part of the Gal4 protein. 

For further reading see e.g. Old and Primrose 5^th edition p. 261.

Both these applications were used in the Pto story (lecture 24 in disease resistance, 2001) - references to the relevant papres there

Papers using this technique in our own work:

• Finnie, C., Andersen, C.H., Borch, J., Gjetting, S., Christensen, A.B., De Boer, A.H., Thordal-Christensen, H. and Collinge, D.B. 14-3-3 Proteins are involved in an epidermis-specific response to the barley powdery mildew fungus. Plant Molecular Biology in press. 
• Feys, B.J., Moisen, L.J., Newman, M.-A., and Parker J.E.  2001. Direct Interaction Between the Arabidopsis Disease Resistance Signaling Proteins, EDS1 and PAD4. EMBO J. 20:5400-5411.
• Jahn, T., Fuglsang, A.T., Olsson, A., Br黱trup, M., Collinge, D.B., Volkmann, D., Sommarin, M., Palmgren M.G. and Larsson, C. (1997) 14-3-3 protein interacts directly with the C- terminal region of the plant plasma membrane H⁺ATPase. The Plant Cell 9: 1805-1814.

Limitations

This technique is prone to limitations and an interaction indicated by Y2H is not proof that this interaction really takes place. Further evidence for an interaction can be obtained by:

• Affinity chromatography followed by protein identification to discover which proteins can bind to a known protein.
• Gel overlay assays which is like a western blot.
• Co-immunoprecipitation
  

  Immunoprecipitation is a technique in which an antibody is used to remove the antigen (e.g. a protein) from a complex mixture - i.e. protein extract. The antiserum binds the protein, the complex precipitates, and the precipitate centrifuged to yield a pellet. The pellet contains the antigen and any other protein (or substance) bound to the antigen. The pellet can be disrupted by SDS or urea, and the contents identified after further purification by e.g.: 

  • western blotting using an antiserum raised against poteintial binding partners.
  • mass spectroscopy
• Various more sophisticated techniques like Spin resonance.

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