Testing for Protein Interactions Between Two Known Proteins

The yeast two-hybrid system can also be used for the analysis of proteinprotein interactions between two known proteins or for determining what protein domains or amino acid residues mediate protein-protein interactions.

To test for protein-protein interactions between two known proteins the MATCHMAKER pGADT7 AD vector (Fig. 2) is used in conjunction with the pGBKT7 DNA-BD vector. The pGADT7 harbors the T7 promoter, a hemagglutinin epitope tag, and an MCS and expresses proteins fused to the GAL4 AD.

The following protocol outlines how to test for protein-protein interactions between two known proteins:

1. Clone the gene(s) of interest (gene X and gene Y) into pGBKT7 and pGADT7 respectively as outlined in Subheading 3.1.2.

Fig. 4. An agarose gel showing Hindlll restriction digest analysis of cDNA library plasmids recovered from 14 different positive clones. Based on their restriction pattern the clones were classified into different groups. Group A, clones 1, 3, 6; Group B, clones 2, 8; Group C, clones 4, 5; Group D, clones 7, 9; Group E, clones 10, 11; Group F, clones 12, 14; Group G, clone 13.

Table 1

List of Most Common False-Positives Identified by Researchers Conducting Yeast Two-Hybrid Screens

Table 1

List of Most Common False-Positives Identified by Researchers Conducting Yeast Two-Hybrid Screens

Protein

Number of times found as a false-positive

Heat shock proteins

16

Ribosomal proteins

14

Cytomchrome oxidase

5

Other mitochondrial proteins"

3

Proteasome subunits

4

Ferritin

4

Transfer-RNA synthase

3

Collagen-related proteins

3

Zinc finger containing proteins

3

Vimentin

2

Inorganic pyrophosphatase

2

Proliferating cell nuclear antigen

2

"Five other mitochondrial proteins are among other categories. (See ref. 6, www.fccc.edu/research/labs/golemis/).

"Five other mitochondrial proteins are among other categories. (See ref. 6, www.fccc.edu/research/labs/golemis/).

2. The clones should then be tested for suitability in interaction studies. Cotransform the bait vector (pGBKT7/Gene X) and prey vector (pGADT7/gene Y) into the reporter strain with the empty AD and DNA-BD vectors, respectively, using the methods described in Subheading 3.1.3., selecting for positive transformants on SD/-Trp/-Leu (see Note 2).

3. Test the autoactivation properties of the bait and prey vectors, possible toxicity problems, and expression of the fusion proteins (see Subheading 3.1.4.).

4. Once the bait and prey vectors have been tested, cotransform the bait (pGBKT7/ gene X) and prey (pGADT7/gene Y) vectors into a yeast reporter strain (see Subheading 3.1.3.).

5. Select yeast colonies from each transformation and test for restoration of His auxotrophy (see Subheading 3.2.3.) and p-galactosidase activity (see Subheading 3.2.4.).

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