In vitro protein interaction detection systems
View Patent ↗Disclosed herein are in vitro assays for the identification of interactions between proteins or other molecules, the identification of transcriptional activator proteins, and the detection of compounds that inhibit protein/protein or protein/compound interactions. Also disclosed herein are in vitro assays for the selection of interacting proteins and transcriptional activator proteins out of libraries.
1. A method for determining whether a protein interacts with a target compound in vitro, said method comprising the steps of:
(a) providing a DNA-protein fusion molecule comprising (i) a double-stranded DNA having a core promoter operably linked to a nucleic acid sequence, and (ii) a protein covalently attached to said DNA;
(b) providing a target compound fusion molecule comprising a target compound covalently attached to a transcription activation domain;
(c) contacting said DNA-protein fusion molecule with said target compound fusion molecule in an in vitro transcription system under conditions that allow said target compound fusion molecule to interact with the protein of said DNA-protein fusion molecule, wherein an interaction recruits RLNA polymerase and triggers transcription of said nucleic acid sequence; and
(d) detecting increased transcription of said nucleic acid sequence, or a portion thereof, in the presence of said target compound fusion molecule as an indication that the protein of said DNA-protein fusion interacts with the target compound of said target compound fusion molecule.
2. A method for determining whether a protein interacts in vitro with a target compound through an adaptor molecule, said method comprising the steps of:
(a) providing a DNA-protein fusion molecule comprising (i) a double-stranded DNA having a core promoter operably linked to a nucleic acid sequence, and (ii) a protein covalently attached to said DNA;
(b) providing a target compound fusion molecule comprising a target compound covalently attached to a transcription activation domain;
(c) contacting said DNA-protein fusion molecule with said target compound fusion molecule in the presence of an adaptor molecule, wherein said adaptor molecule is necessary for an interaction to occur between the protein of said DNA-protein fusion molecule and said target compound fusion molecule, said contacting being carried out in an in vitro transcription system under conditions that allow said DNA-protein fusion molecule, said adaptor molecule, and said target compound fusion molecule to interact, wherein an interaction recruits RNA polymerase and triggers transcription of said nucleic acid sequence; and
(d) detecting increased transcription of said nucleic acid sequence, or a portion thereof, in the presence of said target compound fusion molecule as an indication that the protein of said DNA-protein fusion interacts with the target compound of said target compound fusion molecule in the presence of said adaptor molecule.
3. The method of any one of claims 1 - 2 , wherein said target compound is selected from the group consisting of a protein, a nucleic acid, or a small molecule.
4. The method of any one of claims 1 - 2 , wherein said transcription activation domain is the herpesvirus VP16 polypeptide.
5. The method of any one of claims 1 - 2 , wherein said DNA-protein fusion is characterized by said protein being covalently attached to said DNA 5′ to said core promoter.
6. The method of claim 1 , wherein the nucleic acid sequence comprises an open reading frame encoding said protein.
7. The method of claim 2 , wherein the nucleic acid sequence comprises an open reading frame encoding said protein.