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US 9,435,055 B2Granted
Method and kit for detecting membrane protein-protein interactions
AssigneeHybrigenics Services S.A.S.
InventorsIgor Stagljar, Michael Hottiger, Daniel Auerbach
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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.
Claims34
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.
4 more elements
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.
3 more elements
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.
9 more elements
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).
10 more elements
Dependencies are inferred from the claim text.