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US 6,332,897 B1Granted
Assay methods
AssigneeGlaxo Wellcome Inc.
InventorsMichael Phillip Weiner, Richard Gordon Buckholz
Life of this patent
Estimating the term…
Abstract
The present invention relates to an automated format for the yeast two hybrid assay for protein-protein interactions.
Claims10
A method for detecting an interaction between a first test protein and a second test protein, comprising:
- (a) providing yeast or bacterial host cells of opposite mating type, one or both of which contain a reporter gene which expresses a detectable protein when the reporter gene is activated by an amino acid sequence including a transcriptional activation domain when the transcriptional activation domain is in sufficient proximity to the reporter gene;
- (b) providing a first chimeric gene the first chimeric gene comprising a DNA sequence that encodes a first hybrid protein, the first hybrid protein comprising:
- (i) a DNA-binding domain that recognizes a binding site on the reporter gene in the host cell;
- and
- (ii) a first test protein or fragment thereof that is to be tested for interaction with at least one second test protein or fragment thereof;
- (c) providing a second chimeric gene, the second chimeric gene comprising a DNA sequence that encodes a second hybrid protein, the second hybrid protein comprising:
- (i) the transcriptional activation domain;
- and
- (ii) a second test protein or fragment thereof that is to be tested for interaction with the first test protein or fragment thereof;
- wherein interaction between the first test protein and the second test protein in the host cell causes the transcription activation domain to activate transcription of the reporter gene;
- (d) introducing the second chimeric gene into a host cell of a first mating type such that the second hybrid protein is expressed and subsequently introducing said cells into an arraying means thereby creating a master library plate;
- (e) introducing cells from the master library plate into a second arraying means in liquid medium, thereby creating a mating set;
- (f) introducing the first chimeric gene into a host cell of the opposite mating type of the host cell in step (d) such that the first hybrid protein is expressed and subsequently introducing said cell into the mating set, thereby allowing mating to occur in liquid medium;
- (g) selecting for outgrowth of cells which express the reporter gene, thereby creating a selected mating set;
- (h) removing a portion of the selected mating set to a third arraying means thereby creating a rescue set;
- (i) determining whether the reporter gene has been expressed in cells in the selected mating set;
- and
- (j) analyzing cells from the rescue set which are the same as cells in the selected mating set in which the reporter gene is expressed, wherein detection of expression of the reporter gene in a cell is indicative of an interaction between the first test protein and the second test protein.
15 more elements
A method for detecting an interaction between a first test protein and a second test protein, comprising:
- (a) providing yeast or bacterial host cells of opposite mating type, one or both of which contain a reporter gene which expresses a detectable protein when the reporter gene is activated by an amino acid sequence including a transcriptional activation domain when the transcriptional activation domain is in sufficient proximity to the reporter gene;
- (b) providing a first chimeric gene, the first chimeric gene comprising a DNA sequence that encodes a first hybrid protein, the first hybrid protein comprising:
- (i) a DNA-binding domain that recognizes a binding site on the reporter gene in the host cell;
- and
- (ii) a first test protein or fragment thereof that is to be tested for interaction with at least one second test protein or fragment thereof;
- (c) providing a second chimeric gene, the second chimeric gene comprising a DNA sequence that encodes a second hybrid protein, the second hybrid protein comprising:
- (i) the transcriptional activation domain;
- and
- (ii) a second test protein or fragment thereof that is to be tested for interaction with the first test protein or fragment thereof;
- wherein interaction between the first test protein and the second test protein in the host cell causes the transcription activation domain to activate transcription of the reporter gene;
- (d) introducing the second chimeric gene into a host cell of a first mating type such that the second hybrid protein is expressed and subsequently introducing said cells into an arraying means thereby creating a master library plate;
- (e) introducing cells from the master library plate into a second arraying means in liquid medium, thereby creating a mating set;
- (f) introducing the first chimeric gene into a host cell of the opposite mating type of the host cell in step (d) such that the first hybrid protein is expressed and subsequently introducing said cell into the mating set, thereby allowing mating to occur in liquid medium;
- (g) selecting for outgrowth of cells which express the reporter gene, wherein expression of the reporter gene is indicative of an interaction between the first test protein and the second test protein in that cell.
11 more elements
A method for the selection of a test protein or fragment useful in the detection of protein-protein interactions, said method comprising:
- (a) recoding a yeast activation domain gene, said recoding incorporating DNA controlling elements necessary for E. coli expression, said DNA controlling elements comprising
- (i) a sequence which acts as an E. coli promoter to initiate mRNA transcription;
- (ii) a ribosome binding site and an ATG fMet codon necessary to initiate protein translation;
- (iii) a cloning site composed of one or more restriction sites into which to clone stuffer fragments of DNA that can encode protein fusions to the activation domain;
- and
- (iv) a reporter gene, said reporter gene cloned out-of-frame with respect to the ATG codon;
- (b) cloning stuffer fragments of DNA into the cloning site;
- (c) fusing the stuffer fragments of DNA to the activation domain;
- and
- (d) separating clones that restore the reading frame of the reporter gene wherein a clone that restores the open reading frame is indicative of a test protein.
7 more elements
Dependencies are inferred from the claim text.