His3

LacZ

Select for growth in His- media FIGURE 26.18 Vectors for Two-Hybrid Analysis

Identify blue colonies on X gal

Two different vectors are necessary for two-hybrid analysis. The bait vector has the coding regions for the DBD and for the Bait protein. The prey vector has the coding regions for the AD and for the Prey protein. These two different constructs are expressed in the same yeast cell. If the Bait and Prey interact, the reporter gene is expressed. Two possible reporter systems are shown here. If the yeast His3 gene is used, yeast expressing the reporter gene will be able to make histidine and hence to grow in media without histidine provided. If the lacZ gene from E. coli is used, the yeast cells will turn blue on plates containing X-gal.

Protein Interaction by Co-Immunoprecipitation 737

and a prey plasmid. If the two fusion proteins X and Y interact, the reporter gene is switched on. In yeast, the HIS3 or URA3 genes are usually used. If the reporter gene is not activated, the yeast strain cannot grow unless provided with histidine or uracil respectively. If the reporter gene is turned on the cells can grow on medium without histidine or uracil. Thus the diploid cells from the 6,000 x 6,000 matings are selected on medium lacking the chosen nutrient (Fig. 26.19). Only those combinations where proteins X and Y interact yield viable colonies.

The original two-hybrid system has several limitations. For example, it relies on proteins interacting within the nucleus. Membrane proteins often misfold when localized in the nucleus. Conversely, other proteins are only correctly modified when present in the cytoplasm. Toxic effects and steric problems with very large proteins may also cause some interactions to be missed. Furthermore, many proteins bind RNA and/or rely on small molecules to alter their conformation so promoting proteinprotein interactions.

A variety of modified two-hybrid systems have been developed to deal with these issues. One of the most interesting is the RNA three-hybrid system (Fig. 26.20). In this case, the two proteins (DBD-X and Y-AD) are brought together by an intervening RNA molecule that is bound by both X and Y. This can be used to screen for genes encoding RNA-binding proteins.

Libraries of genes from other organisms can also be used for two-hybrid screening provided they are expressed in yeast. In addition, a two-hybrid screening system (BacterioMatchTM from Stratagene Corporation) has recently been devised for use in the bacterium, E. coli. This uses two tandem reporter genes, bla and lacZ, that encode beta-lactamase (ampicillin resistance) and beta-galactosidase respectively.

Protein Interaction by Co-Immunoprecipitation

In mammalian cells, protein interactions can be identified by co-immunoprecipitation (Fig. 26.21). The gene for the protein of interest is transfected into mammalian cells and expressed. Then the protein synthesized is isolated from the cytoplasm using antibodies. If no antibody is available for the protein of interest, it can be tagged with the FLAG peptide (or some other convenient tag). Then the antibody to the FLAG tag is used to isolate the protein. The protein is isolated under conditions in which any other proteins that interact with the protein of interest stay associated. Protein A from Staphylococcus binds tightly to antibodies and immobilized protein A is therefore used to isolate the antibodies plus any attached proteins. The protein complexes are then separated by electrophoresis on an SDS-PAGE gel to see how many individual proteins are associated in the complex. The identity of the associated proteins can be determined using such techniques as mass spectroscopy or protein sequencing.

Co-immunoprecipitation can confirm protein interactions that were established using the two-hybrid system (Fig. 26.22). If mammalian proteins are found to interact using the two-hybrid system, their interaction must be confirmed in mammalian cells. First, the two proteins of interest are genetically linked to two different tags, such as the FLAG or His6 tags. The two constructs are co-transfected into cultured mammalian cells. Antibody to one of the tags is added to the cell-free extract from one of the cells and incubated. The antibody complex is isolated by binding to beads coated with Protein A, and the fraction is run on SDS-PAGE. The gel is transferred to nitrocellulose and the membrane is probed with separate antibodies to each of the FLAG and His6 tags. If the two proteins of interest interact in mammalian cells, then both proteins will be present on the Western blot.

co-immunoprecipitation Method of identifying protein-protein interaction by using antibodies to a one of the proteins

If two proteins are associated in the cell and one is precipitated by an antibody, the other should accompany it.

Bait library in a MATING TYPE YEAST

Prey library in a MATING TYPE YEAST

Bait library in a MATING TYPE YEAST

Prey library in a MATING TYPE YEAST

Mate by replica plating onto same medium

Diploids form that each contain one bait

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