Methods and materials for increasing expression of recombinant polypeptides
View Patent ↗The present invention provides novel methods and materials for increasing the expression of recombinant polypeptides. Methods and materials of the invention allow increased expression of transcription units that include recombinant DNA sequences which encode polypeptides of interest. The present invention provides expression vectors which contain multiple copies of a transcription unit encoding a polypeptide of interest separated by at least one selective marker gene and methods for sequentially transforming or transfecting host cells with expression vectors to increase transcription unit dosage and expression.
1. A vector or segment thereof comprising multiple copies of a transcription unit separated by at least one selective marker gene wherein the transcription unit encodes a polypeptide,
wherein the vector is linearized with a restriction enzyme, such that the selective marker gene is positioned between at least two transcription unit copies, as [(transcription unit) x -selective marker gene-(transcription unit) x ], and wherein x is 1-4, and
wherein the multiple copies of the transcription unit encode immunoglobulin light and heavy chain polypeptides.
2. A vector or segment thereof comprising multiple copies of a transcription unit separated by at least one selective marker gene wherein the transcription unit encodes a polypeptide,
wherein the vector is linearized with a restriction enzyme, such that the selective marker gene is positioned between at least two transcription unit copies, as [(transcription unit) x -selective marker gene-(transcription unit) x ], and wherein x is 1-4, and
wherein the multiple copies of the transcription unit encode at least the variable regions of immunoglobulin light and heavy chain polypeptides.
3. A vector or segment thereof comprising multiple copies of a transcription unit separated by at least one selective marker gene wherein the transcription unit encodes a polypeptide,
wherein the vector is linearized with a restriction enzyme, such that the selective marker gene is positioned between at least two transcription unit copies, as [(transcription unit) x -selective marker gene-(transcription unit) x ], and wherein x is 1-4, and
wherein the transcription unit encodes a BPI protein product.
4. A vector or segment thereof comprising multiple copies of a transcription unit separated by at least one selective marker gene wherein the transcription unit encodes a polypeptide,
wherein the vector is linearized with a restriction enzyme, such that the selective marker gene is positioned between at least two transcription unit copies, as [(transcription unit) x -selective marker gene-(transcription unit) x ], and wherein x is 1-4, and
wherein the transcription unit encodes a BPI fragment, BPI analog, BPI variant, or BPI-derived peptide.
5. The vector or segment thereof of any one of claims 1 to 4 wherein the number of multiple copies of the transcription unit is two.
6. The vector or segment thereof of any one of claims 1 to 4 wherein each transcription unit is under the control of its own promoter and 3′ untranslated region.
7. The vector or segment thereof of any one of claims 1 to 4 wherein the multiple copies of the transcription unit are in a direct repeat orientation.
8. The vector or segment thereof of any one of claims 1 to 4 wherein the multiple copies of the transcription unit are in an inverted repeat orientation.
9. The vector or segment thereof of any one of claims 1 to 4 wherein the transcription unit comprises an internal ribosome entry site.
10. The vector or segment thereof of any one of claims 1 to 4 wherein the multiple copies of the transcription unit are between 25% and 100% identical.
11. The vector or segment thereof of any one of claims 1 to 4 wherein the multiple copies of the transcription unit are 25% identical.
12. The vector or segment thereof of any one of claims 1 to 4 wherein the multiple copies of the transcription unit are at least 70%, at least 80%, or at least 90% identical.
13. The vector or segment thereof of any one of claims 1 to 4 wherein the multiple copies of the transcription unit are 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical.
14. The vector or segment thereof of any one of claims 1 to 4 wherein the multiple copies of the transcription unit are 100% identical.
15. The vector or segment thereof of any one of claims 1 to 4 wherein the selective marker gene is a gpt, res, neo, his, DHFR, GS, adenosine deaminase (ADA), thymidine kinase (TK), adenine phosphoribosyl transferase (APRT), zeocin resistance, hygromycin resistance or puromycin resistance gene.
16. The vector or segment thereof of claims 3 or 4 wherein the transcription unit encodes rBPI 21 and is under the control of an hCMV promoter and mouse light chain 3′ untranslated region, and wherein the vector further comprises 0, 1 or 2 copies of a human heavy chain enhancer and either a gpt or neo gene.
17. An expression vector or segment thereof according to any one of claims 1 to 4 .
18. A host cell comprising an expression vector or segment thereof according to claim 17 .
19. The host cell according to claim 18 comprising an additional expression vector or segment thereof comprising multiple copies of a transcription unit separated by at least one selective marker gene wherein the transcription unit encodes a polypeptide,
wherein the vector is linearized with a restriction enzyme such that the selective marker gene is positioned between at least two transcription unit copies, as [(transcription unit) x -selective marker gene-(transcription unit) x ], and wherein x is 1-4, said additional expression vector or segment thereof comprising a different selective marker gene.
20. The host cell according to claim 18 wherein the host cell is a eukaryotic cell.
21. The host cell according to claim 18 , wherein the host cell is a plant, insect, mammalian or yeast cell.
22. The host cell according to claim 18 wherein the host cell is a Chinese hamster ovary (CHO) cell or CHO-K1 cell.
23. The host cell according to claim 18 wherein the expression vector or segment thereof is integrated into the host cell chromosome.
24. A stable cell line comprising an expression vector or segment thereof according to claim 17 .
25. The stable cell line according to claim 24 wherein the cells are transformed or transfected by at least one and not more than six expression vectors or segments thereof.
26. The vector or segment of claim 1 , comprising two copies of a transcription unit encoding an immunoglobulin light chain polypeptide and two copies of a transcription unit encoding an immunoglobulin heavy chain polypeptide.
27. A vector or segment thereof comprising multiple copies of a transcription unit each separated by a selective marker gene wherein the transcription unit encodes a polypeptide,
wherein the vector is linearized with a restriction enzyme such that the selective marker gene is positioned between at least two transcription unit copies, as [(transcription unit) x -selective marker gene-(transcription unit) x ], and wherein x is 1-4, and
wherein the multiple copies of the transcription unit encode immunoglobulin light and heavy chain polypeptides.
28. A vector or segment thereof comprising multiple copies of a transcription unit each separated by a selective marker gene wherein the transcription unit encodes a polypeptide,
wherein the vector is linearized with a restriction enzyme such that the selective marker gene is positioned between at least two transcription unit copies, as [transcription unit) x -selective marker gene-(transcription unit) x ], and wherein x is 1-4, and
wherein the multiple copies of the transcription unit encode at least the variable regions of immunoglobulin light and heavy chain polypeptides.
29. A vector or segment thereof comprising multiple copies of a transcription unit each separated by a selective marker gene wherein the transcription unit encodes a polypeptide,
wherein the vector is linearized with a restriction enzyme such that the selective marker gene is positioned between at least two transcription unit copies, as [(transcription unit) x -selective marker gene-(transcription unit) x ], and wherein x is 1-4, and
wherein the transcription unit encodes a BPI protein product.
30. A vector or segment thereof comprising multiple copies of a transcription unit each separated by a selective marker gene wherein the transcription unit encodes a polypeptide,
wherein the vector is linearized with a restriction enzyme such that the selective marker gene is positioned between at least two transcription unit copies, as [(transcription unit) x -selective marker gene-(transcription unit) x ], and wherein x is 1-4, and
wherein the transcription unit encodes a BPI fragment, BPI analog, BPI variant, or BPI-derived peptide.
31. The vector or segment thereof of any one of claims 27 to 30 wherein the number of multiple copies of the transcription unit is two.
32. The vector or segment thereof of any one of claims 27 to 30 wherein each transcription unit is under the control of its own promoter and 3′ untranslated region.
33. The vector or segment thereof of any one of claims 27 to 30 wherein the multiple copies of the transcription unit are in a direct repeat orientation.
34. The vector or segment thereof of any one of claims 27 to 30 wherein the multiple copies of the transcription unit are in an inverted repeat orientation.
35. The vector or segment thereof of any one of claims 27 to 30 wherein the transcription unit comprises an internal ribosome entry site.
36. The vector or segment thereof of any one of claims 27 to 30 wherein the multiple copies of the transcription unit are between 25% and 100% identical.
37. The vector or segment thereof of any one of claims 27 to 30 wherein the multiple copies of the transcription unit are 25% identical.
38. The vector or segment thereof of any one of claims 27 to 30 wherein the multiple copies of the transcription unit are at least 70%, at least 80%, or at least 90% identical.
39. The vector or segment thereof of any one of claims 27 to 30 wherein the multiple copies of the transcription unit are 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical.
40. The vector or segment thereof of any one of claims 27 to 30 wherein the multiple copies of the transcription unit are 100% identical.
41. The vector or segment thereof of any one of claims 27 to 30 wherein the selective marker gene is a gpt, res, neo, his, DHFR, GS, adenosine deaminase (ADA), thymidine kinase (TK), adenine phosphoribosyl transferase (APRT), zeocin resistance, hygromycin resistance or puromycin resistance gene.
42. An expression vector or segment thereof according to any one of claims 27 to 30 .
43. A host cell comprising an expression vector or segment thereof according to claim 42 .
44. The host cell according to claim 43 comprising an additional expression vector or segment thereof comprising multiple copies of a transcription unit each separated by a selective marker gene wherein the transcription unit encodes a polypeptide,
wherein the vector is linearized with a restriction enzyme such that the selective marker gene is positioned between at least two transcription unit copies, as [(transcription unit) x -selective marker gene-(transcription unit) x ], and wherein x is 1-4, said additional expression vector or segment thereof comprising a different selective marker gene.
45. The host cell according to claim 43 wherein the host cell is a eukaryotic cell.
46. The host cell according to claim 43 , wherein the host cell is a plant, insect, mammalian or yeast cell.
47. The host cell according to claim 43 wherein the host cell is a Chinese hamster ovary (CHO) cell or CHO-K1 cell.
48. The host cell according to claim 43 wherein the expression vector or segment thereof is integrated into the host cell chromosome.
49. A stable cell line comprising an expression vector or segment thereof according to claim 42 .
50. The stable cell line according to claim 49 wherein the cells are transformed or transfected by at least one and not more than six expression vectors or segments thereof.