System to detect small molecule/peptide interaction
First Claim
1. A method to determine whether an agent and a target protein interact, said method comprising the steps of:
- contacting an agent/ligand complex comprising a small molecule agent to be tested for binding to a target protein coupled to a ligand that binds a proteinaceous ligand-binding domain witha first fusion protein comprising said target protein and a first complementary portion of a segregable protein; and
a second fusion protein comprising a proteinaceous ligand-binding domain and a second complementary portion of said segregable protein; and
detecting whether the first complementary portion and second complementary portion are brought into proximity.
2 Assignments
0 Petitions
Accused Products
Abstract
Improved methods for determining interactions between peptides or proteins and small molecules are disclosed. The invention methods can be used to screen libraries of either the small molecules or the proteins. In general, the methods comprise contacting an agent/ligand complex consisting essentially of an agent to be tested for binding to a target protein coupled to a ligand capable of binding a proteinaceous ligand-binding domain with a first fusion protein comprising said target protein and a first complementary portion of a segregable protein; and a second fusion protein comprising a proteinaceous ligand-binding domain and a second complementary portion of said segregable protein; and detecting whether the first complementary portion and second complementary portion are brought into proximity.
-
Citations
37 Claims
-
1. A method to determine whether an agent and a target protein interact, said method comprising the steps of:
-
contacting an agent/ligand complex comprising a small molecule agent to be tested for binding to a target protein coupled to a ligand that binds a proteinaceous ligand-binding domain with a first fusion protein comprising said target protein and a first complementary portion of a segregable protein; and a second fusion protein comprising a proteinaceous ligand-binding domain and a second complementary portion of said segregable protein; and detecting whether the first complementary portion and second complementary portion are brought into proximity. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10)
-
-
11. A recombinant host cell in vitro modified to contain:
-
i) a target protein expression unit which comprises a nucleotide sequence encoding a fusion protein comprising a target protein and a first complementary portion of a segregable active protein; and ii) a ligand binding domain expression unit which comprises a nucleotide sequence encoding a fusion protein comprising a ligand-binding domain and a second complementary portion of said segregable active protein which is modified to contain an agent/ligand complex comprising a small molecule agent to be tested for binding to said target protein coupled to a ligand which binds said proteinaceous ligand-binding domain. - View Dependent Claims (12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24)
-
-
25. A method to determine whether a small molecule agent and a target protein interact, said method comprising the steps of:
-
a) incubating a recombinant host cell in vitro which contains i) a detectable marker expression unit which comprises an inducible promoter operably linked to a nucleotide sequence encoding a detectable marker, wherein the expression of said detectable maker is regulated by said inducible promoter; ii) a target protein expression unit which comprises a nucleotide sequence encoding a fusion protein comprising a target protein and a first portion of a transcription activator protein selected from the group consisting of a DNA binding domain and an RNA polymerase activation domain; iii) a ligand binding domain expression unit which comprises a nucleotide sequence encoding a fusion protein comprising a ligand binding domain and a second portion of a transcription activator protein selected from the group consisting of a DNA binding domain and an RNA polymerase activation domain, whichever is not employed in (ii); and iv) a small molecule agent/ligand complex wherein said ligand binds to said ligand-binding domain; under conditions in which said expression units (ii) and (iii) are expressed; b) detecting whether said detectable marker is expressed; and c) determining the level of binding of said agent to said target protein by the level of expression of said detectable marker. - View Dependent Claims (26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37)
-
Specification