# US 10,370,697 B2 · Propeptide-luciferase fusion proteins and methods of use thereof > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,370,697 B2 - Document ID: US10370697B2 - Status: Granted - Kind: B2 - Application number: 15/600,475 - Filed: 2017-05-19 - Published: 2017-09-14 - Granted: 2019-08-06 - Earliest claimed: 2011-11-07 - Assignees: The Broad Institue, Inc.; The General Hospital Corporation; Instituto Carlos Slim De La Salud, A.C. - Applicants: The Broad Institute, Inc.; The General Hospital Corporation; Instituto Carlos Slim De La Salud, A.C. - Inventors: Sean Burns; David Altshuler; Amedeo Vetere - Web page: https://patentgrep.com/patent/US10370697B2 ## CPC - C12Q 1/66: involving luciferase - C07K 14/575: Hormones (derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin C07K14/665, e.g. corticotropin C07K14/695) - C07K 14/605: Glucagons - C07K 14/62: Insulins - C12N 9/0069: {acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)} - C12Q 1/6897: involving reporter genes operably linked to promoters - G01N 33/5008: {for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics} - G01N 33/5035: {on sub-cellular localization} - G01N 33/5073: {Stem cells} - G01N 33/581: {with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)} - C07K 2319/00: Fusion polypeptide - C07K 2319/50: containing protease site - C07K 2319/60: containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP] - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) ## 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. ## Claims ### Claim 1 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. ### Claim 2 (depends on 1) The method of claim 1, wherein the propeptide is selected from the group consisting of a prohormone, a preprohormone, a cytokine precursor and a neuropeptide precursor. ### Claim 3 (depends on 2) The method of claim 2, wherein the preprohormone is preproinsulin and the bioluminescent protein is within the C-peptide component of preproinsulin. ### Claim 4 (depends on 2) The method of claim 2, wherein the preprohormone is preproamylin, wherein the preproamylin has a signal peptide and comprises a mature amylin hormone sequence within preproamylin, wherein the bioluminescent protein is inserted between regions encoding the signal peptide and the mature amylin hormone sequence or at the C-terminus of the mature amylin hormone sequence. ### Claim 5 (depends on 2) The method of claim 2, wherein the preprohormone is preproglucagon comprising a signal peptide, glicentin-related polypeptide (GRPP), glucagon, glucagon-like peptide 1 (GLP-1), and glucagon-like peptide 2 (GLP-2), wherein the bioluminescent protein is inserted between the regions encoding the GRPP and glucagon, glucagon and GLP-1, or GLP-1 and GLP-2. ### Claim 6 (depends on 1) The method of claim 1, wherein the bioluminescent protein comprises less than 200 amino acids. ### Claim 7 (depends on 1) The method of claim 1, wherein the nucleic acid construct comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 5-100. ### Claim 8 (depends on 1) The method of claim 1, wherein the nucleic acid construct encoding the first or second cleavage site or the first and second cleavage sites has a sequence selected from the group consisting of SEQ ID NOs: 101-138. ### Claim 9 (depends on 1) The method of claim 1, wherein the nucleic acid construct further comprises a sequence encoding an additional cleavage site in the fusion protein at the N-terminal or C-terminal end of the bioluminescent protein, or at both the N-terminal and C-terminal ends of the bioluminescent protein. ### Claim 10 (depends on 1) The method of claim 1, wherein the nucleic acid construct further comprises at least 3 additional nucleotides flanking the nucleic acid sequence encoding at least one cleavage site. ### Claim 11 (depends on 1) The method of claim 1, wherein the nucleic acid construct further comprises a promoter. ### Claim 12 (depends on 1) The method of claim 1, wherein the bioluminescent protein is a luciferase, optionally a Gaussia luciferase, optionally wherein the Gaussia luciferase lacks a native signal sequence, optionally wherein the Gaussia luciferase comprises SEQ ID NO: 1. ### Claim 13 (depends on 12) The method of claim 12, wherein the luciferase is a Cypridina luciferase, optionally wherein the Cypridina luciferase lacks a native signal sequence, optionally wherein the Cypridina luciferase comprises SEQ ID NO: 3. ### Claim 14 (depends on 1) The method of claim 1, wherein the cell is selected from the group consisting of an endocrine cell, an immune cell, a neuron, a hepatocyte, a myocyte, a kidney cell, an adipocyte, and an osteocyte. ### Claim 15 (depends on 14) The method of claim 14, wherein the cell is an endocrine cell selected from the group consisting of a beta cell, an alpha cell, an L cell, a K cell, and another endocrine cell. ### Claim 16 (depends on 14) The method of claim 14, wherein the immune cell is selected from the group consisting of a B cell, a T cell, a monocyte, a macrophage, a dendritic cell, a mast cell, and a neutrophil. ### Claim 17 (depends on 1) The method of claim 1, wherein the cell comprises at least two nucleic acid constructs encoding a fusion protein comprising a propeptide linked to a bioluminescent protein, wherein the cell is capable of expressing the encoded fusion proteins and wherein the bioluminescent proteins of the at least two nucleic acid constructs are different. ### Claim 18 (depends on 17) The method of claim 17, wherein the cell comprises a first nucleic acid construct and a second nucleic acid construct, wherein the first nucleic acid construct comprises a Gaussia luciferase and wherein the second nucleic acid construct comprises a Cypridina luciferase. ### Claim 19 (depends on 1) The method of claim 1, wherein the cell further comprises a control nucleic acid construct encoding a control luciferase as an internal reference, wherein the cell is capable of expressing the control luciferase. ### Claim 20 (depends on 1) The method of claim 1, wherein the nucleic acid construct: comprises any one of the nucleotide sequences of SEQ ID NO: 139-330, or encodes any one of the propeptide-bioluminescent fusion protein sequences of SEQ ID NO: 331-395. ### Claim 21 (depends on 1) The method of claim 1, wherein the cell is of the same species as that of the propeptide sequence of the nucleic acid construct. ### Claim 22 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. ### Claim 23 (depends on 22) The method of claim 22, wherein the separating of the population of cells into single cells is performed by a microfluidic device. ### Claim 24 (depends on 22) The method of claim 22, wherein the test compound is a test nucleic acid and further comprising determining the identity of the test nucleic acid using single cell nucleic acid amplification methods. ### Claim 25 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. ### Claim 26 (depends on 25) The method of claim 25, wherein the test compound is a nucleic acid or a small molecule. ### Claim 27 (depends on 26) The method of claim 26, wherein the nucleic acid is an RNAi molecule.