# US 9,435,055 B2 · Method and kit for detecting membrane protein-protein interactions > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 9,435,055 B2 - Document ID: US9435055B2 - Status: Granted - Kind: B2 - Application number: 10/509,507 - Filed: 2003-03-28 - Published: 2005-05-19 - Granted: 2016-09-06 - Earliest claimed: 2002-03-28 - Assignees: Hybrigenics Services S.A.S. - Applicants: Igor Stagljar; Michael Hottiger; Daniel Auerbach - Inventors: Igor Stagljar; Michael Hottiger; Daniel Auerbach - Web page: https://patentgrep.com/patent/US9435055B2 ## CPC - C40B 30/04: by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding - G01N 33/5008: {for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics} - G01N 33/6845: {Methods of identifying protein-protein interactions in protein mixtures} - C40B: COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES (in silico combinatorial libraries of nucleic acids, proteins or peptides G16B35/00; in silico combinatorial chemistry G16C20/60) - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) ## Abstract The present invention is concerned with a method and a kit for detecting an interaction between a first membrane bound test protein or fragment thereof and a second test protein or fragment thereof which is either membrane bound or soluble with an in vivo genetic system based in yeast, bacterial or mammalian cells. The system makes use of the reconstitution of the split ubiquitin protein. ## Claims ### Claim 1 A method for detecting an interaction between a first membrane bound protein or part thereof and a second protein or part thereof, which is either membrane bound or soluble, the method comprising: (a) providing a host cell containing at least one detectable gene (reporter gene) having a binding site for a transcriptional activator, such that the detectable gene expresses a detectable product when the detectable gene is transcriptionally activated; (b) providing, as part of a bait vector, a first chimeric gene under the control of a promoter, said first chimeric gene being expressed in said host cell and coding inter alia for a first membrane protein or part thereof which gene is attached to the DNA-sequence of a first module encoding inter alia a first protein sequence involved in intracellular protein degradation and a transcriptional activator, said first protein or part thereof to be tested whether it can interact with a second protein or part thereof, wherein said bait vector is a plasmid construct; (c) providing, as a part of a prey vector, a second chimeric gene under the control of a promoter that can be expressed in said host cell, the second chimeric gene coding inter alia for a second protein or part thereof which is either membrane bound or soluble and which gene is attached to the DNA sequence of a second module encoding inter alia a second protein sequence involved in intracellular protein degradation, wherein said prey vector is a plasmid construct; (d) introducing the bait vector and the prey vector into the host cell such that an interaction between the expressed first and second proteins and/or their parts can take place, which interaction leads to an interaction of the first protein sequence of the first module and the second protein sequence of the second module which interaction in turn leads to activation of an intracellular protease and proteolytic separation of the transcriptional activator, wherein both the bait vector and the prey vector are maintained episomally; (e) determining whether the detectable gene of the host cell has been activated by the transcriptional activator; and (f) detecting said interaction between said first membrane bound protein or part thereof and said second protein or part thereof. ### Claim 2 (depends on 1) The method according to claim 1, wherein the host cell is a yeast, a bacterium or a mammalian cell. ### Claim 3 (depends on 2) The method according to claim 2, wherein the yeast is Saccharomyces pombe or Saccharomyces cerevisiae. ### Claim 4 (depends on 1) The method according to claim 1, wherein the detectable gene is activated by an activator comprising a short tagging module. ### Claim 5 (depends on 1) The method according to claim 1, wherein the detectable gene is activated by the artificial transcriptional activator protein A-LexA-V16 (PLV). ### Claim 6 (depends on 1) The method according to claim 1, wherein the first protein sequence comprises a C-terminal portion of ubiquitin (Cub) or a mutant thereof (CbM) and the second protein sequence comprises an N-terminal portion of ubiquitin (Nub) or a mutant thereof (NbM). ### Claim 7 (depends on 1) The method according to claim 1, wherein the DNA-sequence coding for the first membrane protein is selected from the group consisting of any bacterial membrane protein, any viral membrane protein, any oncogene-encoded membrane protein, any growth factor receptor or any eukaryotic membrane protein, or parts thereof. ### Claim 8 (depends on 1) The method according to claim 1, wherein the second membrane protein or the soluble protein, or part thereof, is encoded by a plasmid library. ### Claim 9 (depends on 1) The method according to claim 1, wherein the first membrane protein is a soluble protein attached artificially to the membrane via fusion to a signal sequence which encodes a membrane anchor. ### Claim 10 A kit for detecting binding between a first membrane bound protein or part thereof and a second protein or part thereof which is either membrane bound or soluble comprising: (a) a host cell containing at least one detectable gene (reporter gene) having a binding site for a transcriptional activator, such that the detectable gene expresses a detectable product when the detectable gene is transcriptionally activated; (b) a first vector (bait), which is a plasmid construct that is maintained episomally, comprising a first site that can receive a first nucleic acid coding for a first membrane protein or part thereof such that when the first nucleic acid is inserted it becomes attached to the DNA sequence of a first module encoding inter alia a first protein sequence involved in intracellular protein degradation, the first module further comprising a nucleic acid for a transcriptional activator and a promoter; (c) a second vector (prey), which is a plasmid construct that is maintained episomally, comprising a second site that can receive a second nucleic acid coding for a second membrane protein or a soluble protein or part thereof such that when the second nucleic acid is inserted it becomes attached to the DNA sequence of a second module encoding inter alia a sequence protein sequence involved in intracellular protein degradation, wherein the second module further comprises a promoter; and optionally (d) a plasmid library encoding second proteins or parts thereof, wherein binding between said first membrane bound protein or part thereof and said second protein or part thereof is detected. ### Claim 11 (depends on 10) The kit according to claim 10, wherein the host cell is a yeast, a bacterium or a mammalian cell. ### Claim 12 (depends on 11) The kit according to claim 11, wherein the yeast is Saccharomyces pombe or Saccharomyces cerevisiae. ### Claim 13 (depends on 10) The kit according to claim 10, wherein the detectable gene can be activated by an activator comprising a short tagging module. ### Claim 14 (depends on 10) The kit according to claim 10, wherein the detectable gene can be activated by the artificial transcriptional activator protein A-LexA-V16 (PLV). ### Claim 15 (depends on 10) The kit according to claim 10, wherein the first protein sequence contains Cub or CbM and the second protein sequence contains Nub or NbM. ### Claim 16 (depends on 10) The kit according to claim 10, wherein the promoter in (b) is a CYC1 promoter or a CUP1 promoter. ### Claim 17 (depends on 10) The kit according to claim 10, wherein the DNA sequence coding for the first membrane protein is derived from any bacterial membrane protein, any viral membrane protein, any oncogene-encoded membrane protein, any growth factor receptor or any eukaryotic membrane protein, or parts thereof. ### Claim 18 (depends on 10) The kit according to claim 10, wherein the DNA sequence coding for the second protein is contained in a plasmid library. ### Claim 19 A vector comprising the following elements: (a) a selection marker for propagation of the vector in E. coli; (b) an origin of replication which allows propagation of the vector in E. coli; (c) a further selection marker for propagation of the vector in yeast; (d) an origin of replication which allows episomal propagation of the vector in yeast; and (e) an expression cassette comprising the following elements: (i) a promoter element; (ii) a nucleic acid sequence encoding a leader selected from a signal sequence derived from a yeast integral membrane protein and a signal sequence, which confers fatty acid modification; (iii) a nucleic acid sequence encoding Cub or CbM; (iv) a nucleic acid sequence encoding a DNA binding protein; and (v) a nucleic acid sequence encoding a transcriptional activator, wherein said vector is a plasmid. ### Claim 20 (depends on 19) A host cell containing the vector of claim 19. ### Claim 21 (depends on 10) A method of identifying compounds comprising providing the kit of claim 10 and screening compounds for their ability to interfere with protein-protein interaction. ### Claim 22 (depends on 20) A method for providing a compound that can interfere with protein/protein interaction, which method comprises: (a) providing a host cell according to claim 20, the bait and prey polypeptides being selected such that they interact when expressed; (b) incubating the host cell in the presence and absence of the compound(s) to be tested; (c) measuring the difference in reporter gene expression between the incubation containing the compound(s) to be tested and the incubation free of the compound(s) to be tested; and optionally (d) purifying or synthesizing the compound that interferes with protein-protein interaction. ### Claim 23 (depends on 21) The method of claim 21, wherein said compound is a pharmaceutical drug. ### Claim 24 (depends on 1) The method of claim 1, wherein the bait vector is a low copy vector. ### Claim 25 (depends on 24) The method of claim 24, wherein the bait vector is present in 1 to 2 copies per cell. ### Claim 26 (depends on 19) The vector of claim 19, wherein said origin of replication in (d) is a CEN/ARS origin of replication. ### Claim 27 A vector comprising the following elements: (a) a selection marker for propagation of the vector in E. coli; (b) an origin of replication which allows propagation of the vector in E. coli; (c) a further selection marker for propagation of the vector in yeast; (d) an origin of replication which allows episomal propagation of the vector in yeast; and (e) an expression cassette comprising the following elements: (i) a promoter element; (ii) a nucleic acid sequence encoding a leader selected from a signal sequence derived from a yeast integral membrane protein and a signal sequence, which confers fatty acid modification; (iii) a nucleic acid sequence encoding Cub or CbM; (iv) a nucleic acid sequence encoding a DNA binding protein; and (v) a nucleic acid sequence encoding a transcriptional activator, wherein said vector is a plasmid, wherein said origin of replication in (d) is a CEN/ARS origin of replication and wherein the signal sequence encoded in (e)(ii) is: N-MGCTLSAEDKPGGP-C(SEQ ID No. 1). ### Claim 28 (depends on 1) The vector of claim 1, wherein said promoter in (b) is a promoter that confers low level expression. ### Claim 29 (depends on 28) The vector of claim 28, wherein said promoter in (b) is a CYC1 promoter. ### Claim 30 (depends on 28) The vector of claim 28, wherein said promoter in (b) is a CUP1 promoter. ### Claim 31 (depends on 1) The method of claim 1, wherein the bait vector propagates via a CEN/ARS origin of replication. ### Claim 32 (depends on 31) The method of claim 31, wherein the prey vector propagates via a CEN/ARS or 2 micron origin of replication. ### Claim 33 (depends on 10) The kit of claim 10, wherein the first vector propagates via a CEN/ARS origin of replication. ### Claim 34 (depends on 33) The kit of claim 33, wherein the second vector propagates via a CEN/ARS or 2 micron origin of replication.