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US 10,370,697 B2Granted
Propeptide-luciferase fusion proteins and methods of use thereof
AssigneesThe Broad Institue, Inc., The General Hospital Corporation, Instituto Carlos Slim De La Salud, A.C.
InventorsSean Burns, David Altshuler, Amedeo Vetere
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Abstract
The present invention provides nucleic acid constructs that encode fusion peptides comprising a bioluminescent protein and a precursor of a secreted peptide or protein expressed at the cell surface and high throughput screening assays using same.
Claims27
A method for screening for compounds that modulate peptide secretion or localization at the surface of a cell having a cell surface and a secretory pathway comprising:
- a) contacting a cell culture with a test compound, wherein the cell culture comprises a cell comprising a nucleic acid construct encoding a fusion protein comprising a propeptide linked to a bioluminescent protein, wherein the bioluminescent protein is flanked by two cleavage sites, the first of which is positioned 0, 1, 2, 3 or 4 amino acids from the N-terminal end of the bioluminescent protein and the second of which is positioned 0, 1, 2, 3 or 4 amino acids from the C-terminal end of the bioluminescent protein, such that when the fusion protein is expressed by the cell, the fusion protein is transported through the secretory pathway and the bioluminescent protein is cleaved from the propeptide within a secretory vesicle and wherein the cleaved bioluminescent protein is secreted simultaneously with secretion or cell surface localization of the mature form of the propeptide and
- b) determining bioluminescence activity in the cell culture, wherein a difference in bioluminescence from the cell culture that has been contacted with the test compound compared to bioluminescence from a control cell culture indicates that the test compound modulates peptide secretion or localization at the cell surface.
A method for screening for compounds that modulate peptide secretion or localization at the surface of a cell having a cell surface and a secretory pathway comprising:
- a) contacting a population of cells in cell culture media with a test compound, wherein the population of cells comprises a cell comprising a nucleic acid construct encoding a fusion protein comprising a propeptide linked to a bioluminescent protein, wherein the bioluminescent protein is flanked by two cleavage sites, the first of which is positioned 0, 1, 2, 3 or 4 amino acids from the N-terminal end of the bioluminescent protein and the second of which is positioned 0, 1, 2, 3 or 4 amino acids from the C-terminal end of the bioluminescent protein, such that when the fusion protein is expressed by the cell, the fusion protein is transported through the secretory pathway and the bioluminescent protein is cleaved from the propeptide within a secretory vesicle and wherein the cleaved bioluminescent protein is secreted into the cell culture media simultaneously with secretion or cell surface localization of the mature form of the propeptide;
- b) separating the population of cells into single cells;
- and
- c) determining bioluminescence activity of cell culture media of a single cell of the population of cells, wherein a difference in bioluminescence between cell culture media of a single cell that has been contacted with the test compound and cell culture media of a control cell culture indicates that the test compound modulates peptide secretion or localization at the cell surface.
1 more element
A method for screening for compounds that differentiate embryonic stem cells or iPS cells into mature pancreatic beta cells comprising:
- a) contacting a cell culture with a test compound, wherein the cell culture comprises a cell comprising a nucleic acid construct encoding a fusion protein comprising a propeptide linked to a bioluminescent protein, wherein the bioluminescent protein is flanked by two cleavage sites, the first of which is positioned 0, 1, 2, 3 or 4 amino acids from the N-terminal end of the bioluminescent protein and the second of which is positioned 0, 1, 2, 3 or 4 amino acids from the C-terminal end of the bioluminescent protein, such that when the fusion protein is expressed by the cell, the fusion protein is transported through the secretory pathway and the bioluminescent protein is cleaved from the propeptide within a secretory vesicle, and wherein the cleaved bioluminescent protein is secreted simultaneously with secretion or cell surface localization of the mature form of the propeptide and
- b) determining bioluminescence in the cell culture, wherein an increase of bioluminescence from the cell culture that has been contacted with the test compound compared to bioluminescence from a control cell culture indicates that the test compound differentiates embryonic stem cells or iPS cells into mature pancreatic beta cells.
Dependencies are inferred from the claim text.