US 6,673,540 B1 · Cell systems having specific interaction of peptide binding pairs

Abstract

This invention relates to modified host cells which express heterologous fused proteins and methods of screening for test samples having peptide binding activity; wherein the modified host cell comprises: (a) a gene sequence encoding a heterologous fusion protein; the fusion protein comprising a first peptide of a peptide binding pair, or segment of the first peptide, which is joined to either a DNA binding domain or its corresponding transcriptional activation domain of a transcriptional activation protein; (b) a gene sequence encoding a heterologous fusion protein, the fusion protein comprising a second peptide of the peptide binding pair in (a), or a segment thereof, fused to either a DNA binding domain or its corresponding transcriptional activation domain, whichever one is not employed in (a); (c) a reporter gene operatively associated with the transcriptional activation protein, or a portion thereof; (d) optionally, a deletion or mutation in the chromosomal DNA of the host cell for the transcriptional activation protein if present in the selected host cell.

Claims60

Dependent claims

A method of detecting the ability of a test sample to affect the binding interaction of a first peptide and a second peptide of a peptide binding pair that bind through extracellular interaction in their natural environment, comprising:

(i) culturing at least one cell, wherein the cell comprises;
a) a nucleotide sequence encoding a first heterologous fusion protein comprising the first peptide or a segment thereof joined to a transcriptional activation protein DNA binding domain;
b) a nucleotide sequence encoding a second heterologous fusion protein comprising the second peptide or a segment thereof joined to a transcription activation protein transcriptional activation domain;
wherein binding of the first peptide or segment thereof and the second peptide or segment thereof reconstitutes a transcriptional activation protein;
and
c) a reporter gene activated under positive transcriptional control of the reconstituted transcriptional activation protein, wherein expression of the reporter gene produces a selected phenotype;
(ii) incubating a test sample with the cell under conditions suitable to detect the selected phenotype;
and
(iii) detecting the ability of the test sample to affect the binding interaction of the peptide binding pair by determining whether the test sample affects the expression of the reporter gene which produces the selected phenotype.

6 more elements

A rescue screen for detecting the ability of a test sample to affect the binding interaction of a first peptide and a second peptide of a peptide binding pair, comprising:

(i) culturing at least one cell, wherein the cell comprises;
a) a nucleotide sequence encoding a first heterologous fusion protein comprising the first peptide or a segment thereof joined to a transcriptional activation protein DNA binding domain;
b) a nucleotide sequence encoding a second heterologous fusion protein comprising the second peptide or a segment thereof jointed to a transcriptional activation protein transcriptional activation domain;
wherein binding of the first peptide or segment thereof and the second peptide or segment thereof reconstitutes a transcriptional activation protein;
and
c) a reporter gene activated under positive transcriptional control of the reconstituted transcriptional activation protein, wherein expression of the reporter gene prevents exhibition of a selected phenotype;
(ii) incubating a test sample with the cell under conditions suitable to detect the selected phenotype;
and
(iii) detecting the ability of the test sample to affect the binding interaction of the peptide binding pair by determining whether the test sample affects the expression of the reporter gene which prevents exhibition of the selected phenotype.

6 more elements

A rescue screen for determining whether a test sample interacts with a first or second peptide of a peptide binding pair, comprising:

(i) culturing at least one first cell, wherein the first cell comprises;
a) a nucleotide sequence encoding a first heterologous fusion protein comprising the first peptide or a segment thereof joined to a transcriptional activation protein DNA binding domain;
b) a nucleotide sequence encoding a second heterologous fusion protein comprising the second peptide or a segment thereof joined to a transcriptional activation protein transcriptional activation domain;
wherein the nucleotide sequence encoding the first heterologous fusion protein is present in an effective copy number of at least 5 copies per cell and the nucleotide sequence encoding the second heterologous fusion protein is present at a copy number of 1 or 2 per cell;
and
wherein binding of the first peptide or segment thereof and the second peptide or segment thereof reconstitutes a transcriptional activation protein;
and
c) a reporter gene activated under positive transcriptional control of the reconstituted transcriptional activation protein, wherein expression of the reporter gene prevents exhibition of a selected phenotype;
(ii) culturing at least one second cell, wherein the second cell comprises;
a) a nucleotide sequence encoding a first heterologous fusion protein comprising the first peptide or a segment thereof joined to a transcriptional activation protein DNA binding domain;
b) a nucleotide sequence encoding a second heterologous fusion protein comprising the second peptide or a segment thereof joined to a transcriptional activation protein transcriptional activation domain;
wherein the nucleotide sequence encoding the second heterologous fusion protein is present in an effective copy number of at least 5 copies per cell and the nucleotide sequence encoding the first heterologous fusion protein is present at a copy number of 1 or 2 per cell;
and
wherein binding of the first peptide or segment thereof and the second peptide or segment thereof reconstitutes a transcriptional activation protein;
and
c) a reporter gene activated under positive transcriptional control of the reconstituted transcriptional activation protein, wherein expression of the reporter gene prevents exhibition of a selected phenotype;
(iii) incubating a test sample with the first and second cells under conditions suitable to detect the selected phenotype;
(iv) detecting the presence or absence of the selected phenotype produced by the first and second cells;
and
(v) comparing the selected phenotype of the first and second cells, wherein a change in the selected phenotype in one of the cells indicates that the test sample binds to the heterologous fusion protein encoded by the nucleotide sequence present at a copy number of 1 or 2 in that cell exhibiting the change in the selected phenotype, thereby affecting the binding interaction of the peptide binding pair.

17 more elements

A rescue screen for detecting the ability of a test sample to affect the binding interaction of a first and second peptide of a peptide binding pair, comprising:

(i) culturing at least one cell on a selective medium, wherein the cell comprises;
a) a nucleotide sequence encoding a first heterologous fusion protein comprising the first peptide or a segment thereof joined to a transcriptional activation protein DNA binding domain;
b) a nucleotide sequence encoding a second heterologous fusion protein comprising the second peptide or a segment thereof joined to a transcriptional activation protein transcriptional activation domain;
wherein binding of the first peptide or segment thereof and the second peptide or segment thereof reconstitutes a transcriptional activation protein;
c) a reporter gene activated under positive transcriptional control of the reconstituted transcriptional activation protein, wherein when the reporter gene is expressed the cell does not grow on the selective medium;
and
(ii) incubating a test sample with the cell;
and
(iii) detecting the ability of the test sample to affect the binding interaction of the peptide binding pair by determining whether the test sample affects the expression of the reporter gene which prevents growth of the cell on the selective medium.

6 more elements

A rescue screen for determining whether a test sample interacts with a first or second peptide of a peptide binding pair, comprising:

(i) culturing at least one first cell, wherein the first cell comprises;
a) a nucleotide sequence encoding a first heterologous fusion protein comprising the first peptide or a segment thereof joined to a transcriptional activation protein DNA binding domain;
b) a nucleotide sequence encoding a second heterologous fusion protein comprising the second peptide or a segment thereof joined to a transcriptional activation protein transcriptional activation domain;
wherein the nucleotide sequence encoding the first heterologous fusion protein is present in an effective copy number of at least 5 copies per cell and the nucleotide sequence encoding the second heterologous fusion protein is present at a copy number of 1 or 2 per cell;
and
wherein binding of the first peptide or segment thereof and the second peptide or segment thereof reconstitutes a transcriptional activation protein;
and
c) a reporter gene activated under positive transcriptional control of the reconstituted transcriptional activation protein, wherein when the reporter gene is expressed the cell does not grow on the selective medium;
(ii) culturing at least one second cell, wherein the second cell comprises;
a) a nucleotide sequence encoding a first heterologous fusion protein comprising the first peptide or a segment thereof joined to a transcriptional activation protein DNA binding domain;
b) a nucleotide sequence encoding a second heterologous fusion protein comprising the second peptide or a segment thereof joined to a transcriptional activation protein transcriptional activation domain;
wherein the nucleotide sequence encoding the second heterologous fusion protein is present in an effective copy number of at least 5 copies per cell and the nucleotide sequence encoding the first heterologous fusion protein is present at a copy number of 1 or 2 per cell;
and
wherein binding of the first peptide or segment thereof and the second peptide or segment thereof reconstitutes a transcriptional activation protein;
and
c) a reporter gene activated under positive transcriptional control of the reconstituted transcriptional activation protein, wherein when the reporter gene is expressed the cell does not grow on the selective medium;
(iii) incubating a test sample with the first and second cells under conditions suitable to detect the selected phenotype;
(iv) detecting the presence or absence of cell growth by the first and second cells;
and
(v) comparing the growth of the first and second cells, wherein growth of one of the cells indicates that the test sample binds to the heterogeneous fusion protein encoded by the nucleotide sequence present at a copy number of 1 or 2 in the growing cell, thereby affecting the binding interaction of the peptide binding pair.

17 more elements

Dependencies are inferred from the claim text.

Cell systems having specific interaction of peptide binding pairs

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Cell systems having specific interaction of peptide binding pairs

Life of this patent

Abstract

Claims60