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

[0022]In the above aspects of the invention, as well as any aspects herein related to protein-protein interactions, when the first or second protein or protein-binding fragment thereof is a known protein, it is termed the “bait.” The bait can be fused to either the transcriptional inhibitor, in preferred embodiments Gal80 protein, or targeted to a membrane, for example, the cell plasma and vesicular membranes or the mitochondrial outer membrane, by fusion with multicellular organism-based or synthetic cell membrane targeting and anchoring sequences. The protein or protein-binding fragment being tested for protein-protein interactions with the known bait protein is termed the “prey.” When the known bait is fused to the transcriptional inhibitor, e.g., Gal80p, the prey is targeted to one of the above-mentioned membranes. Similarly, the known bait can be targeted to one of the above-mentioned membranes, wherein the prey is fused to transcriptional inhibitor. Further, unknown proteins or protein-binding fragments can be used for fusion to the transcriptional inhibitor and for targeting to the above-mentioned membranes. In such embodiments, both fusions are technically prey; however, the present invention nevertheless enables detection of interactions between two such prey fusions. Interaction between the two fusion proteins traps the transcriptional inhibitor in the cytoplasm, which reduces the amount of transcriptional inhibitor available to suppress transcription of a coding sequence operably-connected to a promoter sensitive to or regulated by the transcriptional inhibitor. For example, interaction between a Gal80p fusion protein and membrane localization sequence-containing fusion protein traps the Gal80p in the cytoplasm. Consequently, less Gal80p is available to bind to any nuclear Gal4p transcription activation domains introduced or otherwise present in the cell from a multicellular organism, which, in turn, activates transcription from any GAL promoters in the nucleus, inducing the expression of GAL reporter genes.

[0032]In the aspects of the invention that relate to NLS detection, any NLSs that are native to the transcriptional inhibitor can be deleted or mutated (i.e., rendered non-functional or of limited function) prior to fusion to putative NLS-containing proteins or protein-binding fragments. In such embodiments, basal-level expression of the gene operably linked to the promoter that is sensitive to or regulated by said transcriptional inhibitor can be greatly increased, thereby enhancing the signal of any non-native NLS introduced in the methods herein. Such an embodiment is particularly useful when the product of the gene operably linked to the promoter that is sensitive to or regulated by said transcriptional inhibitor encodes a product that can convert a non-toxic compound to a cytotoxic or cytostatic compound, or simply encodes a toxic or cytostatic product itself. The present of a NLS decreases the expression of a product that is cytotoxic or cytostatic, thus conferring a growth advantage on those cells that have had a functional NLS fused to the protein or protein-binding fragment.

[0036]In a related ninth aspect, the invention provides a method for detecting a nuclear localization sequence (NLS) in a host cell comprising the steps of introducing into the cell a recombinant expression construct encoding a NLS-containing protein or NLS-containing fragment thereof fused with the amino- or carboxyl-terminus of Gal80p; assaying the cell for expression of a gene operably linked to a promoter that is sensitive to or regulated by Gal80p; and detecting decreased expression of said gene, wherein an NLS is detected thereby. In certain embodiments, one or more of the native Gal80p NLSs are deleted or mutated (i.e., rendered non-functional) prior to fusion to putative NLS-containing protein or protein-binding fragments. Further, in a native-NLS-free background, sequences that act as NLSs or NESs can increase the nuclearity (in the case of a sequence acting as a NLS) or cytoplasmicity (in the case of a sequence acting as a NES). As above, a NES results in increased expression of a gene operably linked to the promoter that is sensitive to or regulated by Gal80p, and a NLS results in decreased expression of the gene operably linked to the promoter that is sensitive to or regulated by Gal80p.

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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