Reverse two-hybrid method

Details Reverse two-hybrid method Reverse two-hybrid method

1994-03-29

1996-06-11

Martinell, James

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving viable micro-organism

43525421, 536 237, C12Q 102, C12N 1581, C12N 1511

Patent

active

055254900

ABSTRACT:
The reverse two-hybrid method has been designed to provide a practical and efficient means of utilizing yeast cell-based assays to screen for molecules that can inhibit protein-protein interactions of interest. Existing two-hybrid systems involve reconstitution in yeast of a transcriptional activator that drives expression of a "reporter" gene such as HIS3 or lacZ. Attempts to utilize these existing systems for drug discovery would necessarily involve screening for molecules that interfere with the transcriptional read-out, and would be subject to detecting any compound that non-specifically interfered with transcription. In addition, since currently used reporter genes encode long-lived proteins, the assay would have to be performed over a lengthy time period to allow for decay of the preexisting reporter proteins. Any compound that would be toxic to yeast over this time period would also score as a "hit". The reverse two-hybrid interaction will avoid both of these pitfalls by driving the expression of a relay gene, such as the GAL80 gene, which encodes a protein that binds to and masks the activation domain of a transcriptional activator, such as Gal4. The reporter genes, which will provide the transcriptional read-out (HIS3 or lacZ), are dependent upon functional Gal4 for expression. Only when the level of Gal80 masking protein is reduced by interfering with the two-hybrid interaction will Gal4 function as a transcriptional activator, providing a positive transcriptional read-out for molecules that inhibit the two-hybrid protein-protein interaction. An important feature of the reverse two-hybrid system is that the basal level and half-life of the relay protein, Gal80, can be fine-tuned to provide maximum sensitivity.

REFERENCES:
"A Novel Genetic System To Detect Protein-protein Interactions", by Fields et al., Nature, vol. 340, Jul. 20, 1989. pp. 245-246.
"A Contingent Replication Assay for the Detection of Protein-protein Interactions in Animal Cells", by Vasavada et al., Proc. Natl. Acad. Sci., vol. 88, pp. 10686-10690, Dec. 1991.
"Mammalian Ras Interacts Directly with the Serine/Threonine Kinase Raf", by Vojtek et al., Cell, vol. 74, pp. 205-214, Jul. 16, 1993.

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