Methods for protein interaction determination
First Claim
1. A method for identifying a plurality of pairs of interacting proteins wherein a pair of interacting proteins comprises a first test protein and a second test protein, wherein the first and second test proteins interact with each other in a cell, the method comprising the steps of:
- a) providing a cDNA library;
b) providing a plurality of a first plasmid comprising a coding sequence for a DNA binding domain of a transcription activator, a first recombinase recognition site, a first selectable marker, a first Type II S restriction site and a first inserted cDNA encoding a first test protein;
c) providing a plurality of a second plasmid comprising a coding sequence for a transcription activation domain of the transcription activator, a second recombinase recognition site, a second selectable marker and a second Type II S restriction site, and a second inserted cDNA encoding a second test protein, wherein the first and second recombinase recognition sites may be identical or distinct and the first and second Type II S restriction sites may be identical or distinct;
d) introducing the first and second plasmids from b) and c) into the same cell;
e) inducing the expression of the recombinase to recombine the first and second introduced plasmids;
f) isolating and digesting the recombined plasmids with a Type II S restriction enzyme to obtain restriction fragments and ligating the restriction fragments to a universal adapter to provide a pool of digested fragments flanked by universal adapter sequences;
g) forming concatamers from the pool of digested fragments and sequencing the concatamers to determine the identity of the plurality of pairs of interacting proteins.
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Abstract
The present invention provides a method for identifying a plurality of pairs of interacting proteins and plasmids for use in the method. The pair of plasmids is adapted for use in a modified two hybrid system wherein wherein each plasmid comprises a recombinase recognition site. The method comprises the steps of providing cDNAs encoding test polypeptides, inserting the cDNAs into the first and second plasmids, recombining the first and second plasmids to obtain recombined plasmids, isolating and digesting the recombined plasmids, ligating the restriction fragments to a universal adapter to provide a pool of digested fragments flanked by a universal adapter, selecting and amplifying desired sequences, forming concatamers from the amplified sequences, and sequencing the concatamers to determine the nucleotide sequences encoding a plurality of pairs of interacting proteins.
13 Citations
21 Claims
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1. A method for identifying a plurality of pairs of interacting proteins wherein a pair of interacting proteins comprises a first test protein and a second test protein, wherein the first and second test proteins interact with each other in a cell, the method comprising the steps of:
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a) providing a cDNA library;
b) providing a plurality of a first plasmid comprising a coding sequence for a DNA binding domain of a transcription activator, a first recombinase recognition site, a first selectable marker, a first Type II S restriction site and a first inserted cDNA encoding a first test protein;
c) providing a plurality of a second plasmid comprising a coding sequence for a transcription activation domain of the transcription activator, a second recombinase recognition site, a second selectable marker and a second Type II S restriction site, and a second inserted cDNA encoding a second test protein, wherein the first and second recombinase recognition sites may be identical or distinct and the first and second Type II S restriction sites may be identical or distinct;
d) introducing the first and second plasmids from b) and c) into the same cell;
e) inducing the expression of the recombinase to recombine the first and second introduced plasmids;
f) isolating and digesting the recombined plasmids with a Type II S restriction enzyme to obtain restriction fragments and ligating the restriction fragments to a universal adapter to provide a pool of digested fragments flanked by universal adapter sequences;
g) forming concatamers from the pool of digested fragments and sequencing the concatamers to determine the identity of the plurality of pairs of interacting proteins. - View Dependent Claims (2, 3, 4, 5, 6, 7)
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8. A plasmid comprising a recombinase recognition site, a cloning site for cloning a cDNA into the plasmid, at least one selectable marker, a Type II S restriction site, and a coding sequence, wherein the coding sequence is selected from the group consisting of:
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a) a coding sequence for a DNA binding domain of a transcription activator such that the DNA binding domain of the transcription activator can be expressed as a fusion protein with the protein encoded by the cDNA; and
b) a coding sequence for a transcription activation domain of a transcription activator such that the DNA transcription activation domain of the transcription activator can be expressed as a fusion protein with the protein encoded by the cDNA. - View Dependent Claims (9, 10, 11, 12, 13, 14)
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15. A kit for determining interacting proteins, wherein the kit comprises:
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a) a first plasmid comprising a coding sequence for a DNA binding domain of a transcription activator, a cloning site for cloning a first cDNA into the first plasmid such that the DNA binding domain of the transcription activator can be expressed as a fusion protein with the protein encoded by the first cDNA, a first recombinase recognition site, a first selectable marker, and a first Type II S restriction site; and
b) a second plasmid comprising a coding sequence for a transcription activation domain of the transcription activator, the cloning site for cloning a second cDNA into the second plasmid such that the transcription activation domain of the transcription activator can be expressed as a fusion protein with the protein encoded by the second cDNA, a second recombinase recognition site, a second selectable marker, and a second Type II S restriction site, wherein the first and second recombinase recognition sites may be identical or distinct and the first and second Type II S restriction sites may be identical or distinct. - View Dependent Claims (16, 17, 18, 19, 20, 21)
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Specification