Light-emitting fusion proteins and diagnostic and therapeutic methods therefor
First Claim
1. A non-invasive method for detecting the localization of an entity in a subject, comprising:
- a) administering to said subject a light-generating fusion protein, or a cell expressing said light-generating fusion protein, said light-generating fusion protein comprising i) a ligand binding site and ii) a light-generating polypeptide moiety, wherein the light generation of said light-generating fusion protein changes upon binding of a ligand at said ligand binding site, said ligand binding site recognizing a ligand which is on said entity or a produced by said entity;
b) allowing for co-localization of said light-generating fusion protein or cell and an entity, wherein contact between said ligand binding site and said ligand causes a modification of a colinear effector site which alters the light generation of said light-generating polypeptide moiety; and
c) imaging said localized light-generating fusion protein, thereby localizing an entity in a subject.
4 Assignments
0 Petitions
Accused Products
Abstract
Light-generating fusion proteins having a ligand binding site and a light-generating polypeptide moiety and their use as diagnostics, in drug screening and discovery, and as therapeutics, are disclosed. The light-generating fusion protein has a feature where the bioluminescence of the polypeptide moiety changes upon binding of a ligand at the ligand binding site. The ligand may be, for example, an enzyme present in an environment only under certain conditions, e.g., ubiquitin ligase in a hypoxic state, such that the light-generating fusion protein is “turned on” only under such conditions.
-
Citations
65 Claims
-
1. A non-invasive method for detecting the localization of an entity in a subject, comprising:
-
a) administering to said subject a light-generating fusion protein, or a cell expressing said light-generating fusion protein, said light-generating fusion protein comprising i) a ligand binding site and ii) a light-generating polypeptide moiety, wherein the light generation of said light-generating fusion protein changes upon binding of a ligand at said ligand binding site, said ligand binding site recognizing a ligand which is on said entity or a produced by said entity;
b) allowing for co-localization of said light-generating fusion protein or cell and an entity, wherein contact between said ligand binding site and said ligand causes a modification of a colinear effector site which alters the light generation of said light-generating polypeptide moiety; and
c) imaging said localized light-generating fusion protein, thereby localizing an entity in a subject. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19)
-
-
20. A method of imaging hypoxic tissue in a mammalian subject, comprising:
-
a) administering to a subject a light-generating fusion protein, or a cell expressing said light-generating fusion protein, comprising an ubiquitin ligase binding site and a light-generating polypeptide moiety, wherein the light generation of said light-generating fusion protein changes upon binding of a ubiquitin ligase at said ubiquitin ligase binding site, said ubiquitin ligase binding site recognizing a ubiquitin ligase present in hypoxic conditions in hypoxic tissue;
b) allowing for localization of said light-generating fusion protein or cell in hypoxic tissue in said subject, wherein contact between said ubiquitin ligase binding site and a ubiquitin ligase causes a modification of a colinear effector site which alters the light generation of said light-generating polypeptide moiety; and
;
c) measuring luminescence of said localized light-generating fusion protein and imaging said localized light-generating fusion protein, thereby imaging said hypoxic tissue. - View Dependent Claims (21, 22, 23, 24, 25, 26, 27, 28)
-
-
29. A method of measuring prolyl hydroxylase activity, comprising the steps of:
-
a) contacting a test sample with a light-generating fusion protein comprising an HIF1α
polypeptide moiety having binding character for prolyl hydroxylase and a light-generating polypeptide moiety, wherein the light generation of said light-generating polypeptide moiety changes upon binding of a prolyl hydroxylase to said HIF1α
polypeptide moiety; and
b) determining prolyl hydroxylase activity by measuring the luminescence of said test sample.
-
-
30. A method of monitoring the activity of an enzyme of interest, comprising:
-
a) exposing, to a fusion protein, a genetically manipulated cell or transgenic non-human mammal containing a recombinant DNA molecule comprising a binding site for a heterologous DNA binding domain upstream of a reporter gene, said reporter gene encoding a light emitting protein;
said fusion protein comprising an enzyme fused to a heterologous DNA binding domain; and
b) monitoring the production of the light emitting protein in the presence of substrate for the enzyme. - View Dependent Claims (31, 32, 33, 34, 35)
-
- 36. A light-generating fusion protein comprising a ligand binding site and a light-generating polypeptide moiety, whereupon ligand binding to said ligand binding site alters the luminescence of said light-generating polypeptide moiety without altering the phosphorylational state of said fusion protein.
-
42. A light-generating fusion protein comprising a ligand binding site and a light-generating polypeptide moiety, whereupon ligand binding to said ligand binding site alters the luminescence and phosphorylational state of said light-generating polypeptide moiety, whereby said alteration in the phosphorylational state of said light-generating fusion protein does not alter the light generation of said light-generating protein.
- 43. A light-generating fusion protein comprising a ubiquitin ligase binding site and a light-generating polypeptide moiety, wherein the light generation of said light-generating fusion protein changes upon binding of a ubiquitin ligase at said ubiquitin ligase binding site.
-
45. A light-generating fusion protein comprising a HIF1α
- polypeptide moiety and a light-generating polypeptide moiety wherein the light generation of said light-generating fusion protein changes upon binding of prolyl hydroxylase to said HIF1α
polypeptide moiety. - View Dependent Claims (46)
- polypeptide moiety and a light-generating polypeptide moiety wherein the light generation of said light-generating fusion protein changes upon binding of prolyl hydroxylase to said HIF1α
-
47. A fusion protein comprising a HIF1α
- polypeptide moiety and a suicide protein polypeptide moiety.
-
48. A fusion protein comprising a HIF1α
- polypeptide moiety, a suicide protein polypeptide moiety, and a light-generating polypeptide moiety, wherein the light generation of said light-generating fusion protein changes upon binding of prolyl hydroxylase to said HIF1α
polypeptide moiety.
- polypeptide moiety, a suicide protein polypeptide moiety, and a light-generating polypeptide moiety, wherein the light generation of said light-generating fusion protein changes upon binding of prolyl hydroxylase to said HIF1α
-
49. A fusion protein comprising a protease binding site and a light-generating polypeptide moiety wherein the light generation of said light-generating fusion protein changes upon binding of a protease to said protease binding site.
-
50. A light-generating fusion protein comprising a protein dimerization domain and a light-generating polypeptide moiety, wherein the light generation of said light-generating fusion protein changes upon ligand binding to said ligand binding site effected via a secondary protein which mediates said ligand binding.
-
51. A non-invasive method for detecting cancerous tissue in a subject, comprising:
-
a) administering to a subject a light-generating fusion protein, or a cell expressing said light-generating fusion protein, comprising a cyclin/cdk binding site and a light-generating polypeptide moiety, wherein the light generation of said light-generating fusion protein changes upon binding of a cyclin at said cyclin binding site, said cyclin binding indicative of cancerous tissue;
b) allowing for localization of said light-generating fusion protein or cell in cancerous tissue in said subject, wherein contact between said cyclin binding site and a cyclin causes a modification of a colinear effector site which alters the light generation of said light-generating polypeptide moiety; and
;
c) measuring luminescence from said localized light-generating fusion protein and imaging said localized light-generating fusion protein, thereby detecting cancerous tissue in said subject. - View Dependent Claims (52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65)
-
Specification