Constructs and cells for production of small peptides
The present invention features a nucleic acid construct for expressing a product, e.g., a small peptide such as GLP-1. The construct includes a nucleic acid sequence encoding a signal peptide and a nucleic acid sequence which encodes the pro-region of somatostatin or a functional fragment thereof. The construct can further include a sequence encoding the small peptide or the construct can be used to express an endogenous genomic sequence encoding the small peptide. The present invention further features genetically engineered cells, and methods of using such constructs or cells.
1. A nucleic acid construct for expression of a glucagon-like peptide-1 (GLP-1) or a variant thereof, comprising:
a nucleic acid sequence encoding a signal peptide from the pre-region of somatostatin;
a nucleic acid sequence encoding the pro-region of a somatostatin, or a functional fragment or a variant of the pro-region of a somatostatin wherein the functional fragment or variant differs from the wild-type amino acid sequence by at least 1 but not more than 5 amino acid residues and is sufficient to promote secretion from a non-endocrine cell; and
a nucleic acid sequence encoding a GLP-1 or a variant thereof.
2. The construct of claim 1 , wherein the nucleic acid sequence encoding the signal peptide comprises the pre-region of a somatostatin.
3. The construct of claim 1 , wherein the construct further comprises a nucleotide sequence encoding a cleavage site between the sequence encoding the pro-region or functional fragment or variant thereof and the sequence encoding the GLP-1 or a variant thereof.
4. The construct of claim 3 , wherein the cleavage site is a multibasic, dibasic or monobasic cleavage site.
5. The construct of claim 3 , wherein the cleavage site is an endoprotease cleavage site.
6. The construct of claim 5 , wherein the cleavage site is recognized by a pro-protein convertase.
7. The construct of claim 6 , wherein the pro-protein convertase is furin, substilisin-related pro-protein convertase, PC1, PC2, PC6 or PC7.
8. The construct of claim 1 , further comprising at least one regulatory sequence.
9. The construct of claim 1 , wherein the variant is selected from the group consisting of: GLP-1 (7-34), GLP-1 (7-35), GLP-1 (7-36), GLP-1 (7-37), Gln 9 -GLP-1 (7-37), Thr 16 -Lys 18 -GLP-1 (7-37), Lys 18 -GLP-1 (7-37), Gly 8 -GLP-1, Met 16 -Met 20 -GLP-1 (7-37).
10. A method of making a cell capable of secreting a GLP-1 or a variant thereof, comprising:
providing a non-endocrine cell; and
introducing into the cell a nucleic acid construct of claim 1 or 3 to thereby obtain a cell capable of expressing the GLP-1 or variant thereof.
11. The method of claim 10 , wherein the cell is a primary cell.
12. The method of claim 10 , wherein the cell is a secondary cell.
13. The method of claim 10 , wherein the cell is a mammalian cell.
14. The method of claim 10 , wherein the sequence encoding the signal peptide comprises the pre-region of a somatostatin.
15. A nucleic acid construct for expression of GLP-1, comprising: a nucleic acid sequence encoding a fusion protein comprising a signal peptide from the pre-region of somatostatin; the pro-region of a somatostatin; and GLP-1.
16. A nucleic acid construct for expression of GLP-1 comprising: a nucleic acid sequence encoding a fusion protein comprising the prepro-region of somatostatin and GLP-1.
17. An isolated non-endocrine cell comprising a nucleic acid sequence that encodes a fusion protein that comprises (a) a signal peptide from the pre-region of somatostatin, (b) a pro-region of a somatostatin or a functional fragment or a variant of the pro-region of a somatostatin wherein the functional fragment or variant differs from the wild-type amino acid sequence by at least 1 but not more than 5 amino acid residues and is sufficient to promote secretion from a non-endocrine cell, and (c) a glucagon-like peptide-1 (GLP-1) or a variant thereof, the cell being capable of secreting the GLP-1 or variant thereof.
18. The cell of claim 17 , wherein the encoded fusion protein further comprises a cleavage site between the pro-region or functional fragment or variant thereof and the GLP-1 or variant thereof.
19. The cell of claim 18 , wherein the cleavage site is a multibasic, dibasic or monobasic cleavage site.
20. The cell of claim 18 , wherein the cleavage site is an endoprotease cleavage site.
21. The cell of claim 20 , wherein the cleavage site is recognized by a pro-protein convertase.
22. The cell of claim 21 , wherein the pro-protein convertase is furin, PACE4, substilisin-related pro-protein convertase, PC1, PC2, PC6 or PC7.
23. The cell of claim 18 , wherein the cleavage site is a blood coagulation factor cleavage site.
24. The cell of claim 17 , wherein the cell is capable of expressing the GLP-1 or variant thereof in mature form without the signal peptide and pro-region of somatostatin or functional fragment or variant thereof.
25. The cell of claim 17 , wherein the cell is a primary cell.
26. The cell of claim 17 , wherein the cell is a secondary cell.
27. The cell of claim 17 , wherein the cell is a mammalian cell.
28. The cell of claim 27 , wherein the cell is a human cell.
29. The cell of claim 27 , wherein the cell is a fibroblast or a myoblast.
30. The cell of claim 17 , wherein the cell is one in which somatostatin is not normally expressed.
31. The cell of claim 17 , wherein the nucleic acid sequence that encodes the fusion protein is operably linked to at least one regulatory sequence sufficient for expression of the fusion protein in the cell.
32. A method of making a GLP-1 or a variant thereof comprising culturing the cell of claim 17 to thereby obtain GLP-1 or a variant thereof.
33. The method of claim 32 , wherein the GLP-1 or variant thereof is obtained in mature form without the signal peptide and pro-region of somatostatin or functional fragment or variant thereof.
34. The method of claim 32 , wherein the GLP-1 or variant thereof is obtained as part of a fusion peptide which further comprises the pro-region of somatostatin or the functional fragment or variant thereof.
35. The cell of claim 17 , wherein the variant is selected from the group consisting of: GLP-1 (7-34), GLP-1 (7-35), GLP-1 (7-36), GLP-1 (7-37), Gln 9 -GLP-1 (7-37), Thr 16 -Lys 18 -GLP-1 (7-37), Lys 18 -GLP-1 (7-37), Gly 8 -GLP-1, Met 16 -Met 20 -GLP-1 (7-37).
36. An isolated non-endocrine, mammalian cell comprising a nucleic acid sequence encoding a fusion protein comprising: a signal peptide from the pre-region of somatostatin, the pro-region of somatostatin, and a GLP-1, wherein the cell secretes the GLP-1.
37. The cell of claim 36 , wherein the cell is a human cell.
38. The cell of claim 36 , wherein the cell is a fibroblast.
39. An isolated non-endocrine, human cell comprising a nucleic acid sequence encoding a fusion protein comprising: the prepro-region of somatostatin and GLP-1, wherein the cell secretes GLP-1.
40. An isolated fibroblast comprising a nucleic acid sequence encoding a fusion protein comprising: the prepro-region of somatostatin and GLP-1 or a variant thereof, wherein the variant is selected from the group consisting of: GLP-1 (7-34), GLP-1 (7-35), GLP-1 (7-36), GLP-1 (7-37), Gln 9 -GLP-1 (7-37), Thr 16 -Lys 18 -GLP-1 (7-37), Lys 18 -GLP-1 (7-37), Gly 8 -GLP-1, Met 16 -Met 20 -GLP-1 (7-37), and the cell secretes the GLP-1 or variant thereof.
41. A method of making GLP-1 or a variant thereof comprising culturing the fibroblast of claim 40 to thereby obtain the GLP-1 or variant thereof.