Detection of protein interactions
First Claim
Patent Images
1. A method for identifying nucleotide sequences encoding interacting polypeptide sequences, the method comprising:
- providing a host cell containing a reporter gene operably linked to a transcriptional regulatory sequence which includes a binding site for a DNA-binding domain;
introducing into the host cell a first chimeric gene encoding a first fusion protein, wherein the first fusion protein comprises a first polypeptide sequence and a DNA binding domain, and wherein the first chimeric gene comprises a first nucleotide sequence encoding the first polypeptide sequence;
introducing into the host cell a second chimeric gene encoding a second fusion protein, wherein the second fusion protein comprises a second polypeptide sequence and an activation tag, and wherein the second chimeric gene comprises a second nucleotide sequence encoding the second polypeptide sequence;
culturing the host cell for a time sufficient to allow an interaction of the first fusion protein and the second fusion protein, wherein the interaction results in a measurable change in expression of the reporter gene;
selecting the host cell based upon the measurable change in expression of the reporter gene; and
sequencing the first nucleotide sequence and the second nucleotide sequence, to thereby identify nucleotide sequences encoding interacting polypeptide sequences.
0 Assignments
0 Petitions
Accused Products
Abstract
The present invention provides methods for determining the sequence of nucleic acids encoding interacting polypeptide sequences. Two hybrid assays are carried out to select host cells containing nucleotide sequences encoding interacting proteins. The identity of the nucleotide sequences is determined by isolating the nucleotide sequences from the selected host cells and carrying out sequencing reactions on the nucleotide sequences.
-
Citations
83 Claims
-
1. A method for identifying nucleotide sequences encoding interacting polypeptide sequences, the method comprising:
-
providing a host cell containing a reporter gene operably linked to a transcriptional regulatory sequence which includes a binding site for a DNA-binding domain;
introducing into the host cell a first chimeric gene encoding a first fusion protein, wherein the first fusion protein comprises a first polypeptide sequence and a DNA binding domain, and wherein the first chimeric gene comprises a first nucleotide sequence encoding the first polypeptide sequence;
introducing into the host cell a second chimeric gene encoding a second fusion protein, wherein the second fusion protein comprises a second polypeptide sequence and an activation tag, and wherein the second chimeric gene comprises a second nucleotide sequence encoding the second polypeptide sequence;
culturing the host cell for a time sufficient to allow an interaction of the first fusion protein and the second fusion protein, wherein the interaction results in a measurable change in expression of the reporter gene;
selecting the host cell based upon the measurable change in expression of the reporter gene; and
sequencing the first nucleotide sequence and the second nucleotide sequence, to thereby identify nucleotide sequences encoding interacting polypeptide sequences. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 77, 78, 79, 80, 81)
-
-
24. A method for identifying nucleotide sequences encoding interacting polypeptide sequences, the method comprising:
-
providing a cell population comprising a plurality of host cells, wherein each of the plurality of host cells contains a reporter gene operably linked to a transcriptional regulatory sequence which includes a binding site for a DNA-binding domain;
introducing into each of the plurality of host cells a first chimeric gene encoding a first fusion protein, wherein the first fusion protein comprises a first polypeptide sequence and a DNA binding domain, wherein the first chimeric gene comprises a first nucleotide sequence which encodes the first polypeptide sequence, and wherein the first nucleotide sequence is different in the first chimeric gene introduced into each of the plurality of host cells;
introducing into each of the plurality of host cells a second chimeric gene encoding a second fusion protein, wherein the second fusion protein comprises a second polypeptide sequence and an activation tag, wherein the second chimeric gene comprises a second nucleotide sequence which encodes the second polypeptide sequence, and wherein the second nucleotide sequence is different in the second chimeric gene introduced into each of the plurality of host cells;
culturing the plurality of host cells for a time sufficient to allow an interaction of the first fusion protein and the second fusion protein, wherein the interaction results in a measurable change in expression of the reporter gene;
selecting the plurality of host cells based upon the measurable change in expression of the reporter gene; and
sequencing the first nucleotide sequence and the second nucleotide sequence contained in each of the plurality of host cells, to thereby identify nucleotide sequences encoding interacting polypeptide sequences.
-
-
70. A method for identifying nucleotide sequences encoding interacting polypeptide sequences, the method comprising:
-
providing a cell population comprising 100,000 bacterial host cells, wherein each of the 100,000 bacterial host cells contains a reporter gene operably linked to a transcriptional regulatory sequence which includes a binding site for a DNA-binding domain;
introducing into each of the 100,000 bacterial host cells a first chimeric gene encoding a first fusion protein, wherein the first fusion protein comprises a first polypeptide sequence and a DNA-binding domain, wherein the first chimeric gene comprises a first nucleotide sequence which encodes the first polypeptide sequence, and wherein the first nucleotide sequence is different in the first chimeric gene introduced into each of the 100,000 bacterial host cells;
introducing into each of the 100,000 bacterial host cells a second chimeric gene encoding a second fusion protein, wherein the second fusion protein comprises a second polypeptide sequence and an activation tag, wherein the second chimeric gene comprises a second nucleotide sequence which encodes the second polypeptide sequence, and wherein the second nucleotide sequence is different in the second chimeric gene introduced into each of the 100,000 bacterial host cells;
culturing the 100,000 bacterial host cells for a time sufficient to allow an interaction of the first fusion protein and the second fusion protein, if present, wherein the interaction results in a measurable change in expression of the reporter gene;
selecting from the 100,000 bacterial host cells those cells that exhibit the measurable change in expression of the reporter gene, to thereby result in selected bacterial host cells; and
sequencing the first nucleotide sequence and the second nucleotide sequence contained in selected bacterial host cells, to thereby identify nucleotide sequences encoding interacting polypeptide sequences.
-
-
71. A method for identifying nucleotide sequences encoding interacting polypeptide sequences, the method comprising:
-
providing a cell population comprising a plurality of host cells, wherein each of the plurality of host cells contains a reporter gene operably linked to a transcriptional regulatory sequence which includes a binding site for a DNA-binding domain;
introducing into each of the plurality of host cells a first chimeric gene encoding a first fusion protein, wherein the first fusion protein comprises a first polypeptide sequence and a DNA binding domain, and wherein the first chimeric gene comprises a first nucleotide sequence encoding the first polypeptide sequence;
introducing into each of the plurality of host cells a second chimeric gene encoding a second fusion protein, wherein the second fusion protein comprises a second polypeptide sequence and an activation tag, wherein the second chimeric gene comprises a second nucleotide sequence which encodes the second polypeptide sequence, and wherein the second nucleotide sequence is different in the second chimeric gene introduced into each of the plurality of host cells;
culturing the plurality of host cells for a time sufficient to allow an interaction of the first fusion protein and the second fusion protein, if present, wherein the interaction results in a measurable change in expression of the reporter gene;
selecting from the plurality of host cells those cells that exhibit the measurable change in expression of the reporter gene, to thereby result in selected host cells;
purifying the second nucleotide sequence from each of the selected host cells, wherein the purification is carried out by an automated process in compartments of a multi-compartment device; and
sequencing the second nucleotide sequence from each of the selected host cells, to thereby identify nucleotide sequences encoding interacting polypeptide sequences. - View Dependent Claims (72, 73, 82)
-
-
74. A method for identifying nucleotide sequences encoding interacting polypeptide sequences, the method comprising:
-
providing a cell population comprising a plurality of host cells, wherein each of the plurality of host cells contains a reporter gene operably linked to a transcriptional regulatory sequence which includes a binding site for a DNA-binding domain;
introducing into each of the plurality of host cells a first chimeric gene encoding a first fusion protein, wherein the first fusion protein comprises a first polypeptide sequence and a DNA binding domain, wherein the first chimeric gene comprises a first nucleotide sequence which encodes the first polypeptide sequence, and wherein the first nucleotide sequence is different in the first chimeric gene introduced into each of the plurality of host cells;
introducing into each of the plurality of host cells a second chimeric gene encoding a second fusion protein, wherein the second fusion protein comprises a second polypeptide sequence and an activation tag, and wherein the second chimeric gene comprises a second nucleotide sequence encoding the second polypeptide sequence;
culturing the plurality of host cells for a time sufficient to allow an interaction of the first fusion protein and the second fusion protein, if present, wherein the interaction results in a measurable change in expression of the reporter gene;
selecting from the plurality of host cells those cells that exhibit the measurable change in expression of the reporter gene, to thereby result in selected host cells;
purifying the first nucleotide sequence from each of the selected host cells, wherein the purification is carried out by an automated process in compartments of a multi-compartment device; and
sequencing the first nucleotide sequence from each of the selected host cells, to thereby identify nucleotide sequences encoding interacting polypeptide sequences. - View Dependent Claims (75, 76, 83)
-
Specification