MGAL: A GAL Gene Switch-Based Suite of Methods for Protein Analyses and Protein Expression in Multicellular Organisms and Cells Therefrom

Abstract

The invention provides methods for detecting and analyzing protein-protein interactions and agonists and antagonists thereof, detecting and analyzing protein sequences, and regulatable gene expression in multicellular organisms or cells therefrom.

Description

[0001]This application claims priority to U.S. provisional application Ser. No. 60 / 390,872 filed Jun. 20, 2002.[0002]This application was supported by a grant from the National Institutes of Health, No. RO1 GM27925-22. The government may have certain rights in this invention. All patents, published patent applications and other references cited in the specification are incorporated herein in their entirety.BACKGROUND OF THE INVENTION[0003]1. Field of the Invention[0004]This invention relates to methods for regulatable gene expression, detection and analysis of protein sequences, and detection and analysis of protein-protein interactions and agonists and antagonists thereof in multicellular organisms or cells therefrom[0005]This invention relates to interactions between biological molecules, particularly proteins, and methods for detecting and quantifying such interactions. The invention is particularly related to detection of protein-protein interactions that occur in the cellular c...

AI Technical Summary

Benefits of technology

The invention provides methods for detecting protein-protein interactions in a cell of a multicellular organism. These methods involve introducing into a cell a first recombinant expression construct encoding a first protein or protein-binding fragment thereof fused with the amino- or carboxyl-terminus of Gal80p, and a second recombinant expression construct encoding a second protein or protein-binding fragment thereof fused with a cytoplasm localization sequence. When the two proteins interact, Gal80p is localized to the cytoplasm, resulting in increased transcription of a gene operably linked to a promoter that is sensitive to or regulated by Gal80p. The methods can be used to detect protein-protein interactions in a host cell cytoplasm, and can also be used to detect agonists and antagonists of transcription factors. The invention also provides methods for detecting protein-protein interactions in the cytoplasm of a cell using a library of cDNAs encoding protein-binding fragments.

Problems solved by technology

Unlike in yeast, there are very few existing methods for detecting and analyzing protein-protein interactions and agonists and antagonists thereof or regulatable gene expression in multicellular organisms or cells therefrom.

Moreover, existing methods suffer from serious limitations.

This system has numerous significant problems, among them the heat shock method of induction and the extended persistence of the Gal80 protein, which can remain in the cells for as long as 48 hours after recombination excision of the GAL80 gene sequences.

One serious limitation of two-hybrid methods in multicellular organisms or cells therefrom is they are not applicable for a very large number of proteins including, but not limited to, most transcriptional activators, transcriptional repressors, RNA polymerase II components, components of the general (basal) transcription machinery, and proteins that are associated with chromatin or participate in chromatin remodeling.

This limitation stems from the existing two-hybrid methods' detection of protein-protein interaction by activating reporter genes through either direct binding to the RNA polymerase or through binding to other proteins that in turn bind to an RNA polymerase subunit.

Another serious limitation of existing methods is that they require that the relevant protein-protein interactions and the detection thereof take place in the nucleus at the reporter gene promoter.

Therefore, existing methods in the art requiring nuclear localization of both interacting proteins are not useful for detecting a significant fraction of protein-protein interactions that normally occur within the cell.

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