Recombinant method for making RSV reverse transcriptases and mutants thereof
View Patent ↗A method of producing a Rous Sarcoma Virus reverse transcriptase (RSV RT) by expressing one orniore nucleic acid sequences encoding one or more subunits of RSV RT in a eukaryotic host cell and culturing the host cell under conditions sufficient to produce the recombinant RSV RT. The resulting RSV RT has a specific activity of between about 30,000 and 150,000 units per milligram and is suitable for methods including RT-polyinerase chain reaction (RT-PCR).
1. A method of producing a Rous Sarcoma Virus (RSV) reverse transcriptase having a RNA-dependent DNA polymerase specific activity of 30,000 units per milligram to 150,000 units per milligram, said method comprising
(a) obtaining a eukaryotic host cell comprising one or more nucleic acid sequences encoding one or more subunits of RSV reverse transcriptase;
(b) culturing said host cell under conditions sufficient to produce said RSV reverse transcriptase; and
(c) isolating or purifying said reverse trascriptase thereby obtaining an RSV reverse transcriptase having a RNA-dependent DNA polymeraso specific activity of 30,000 units per milligram to 150,000 units per milligram.
2. The method of claim 1 , wherein said subunits are α subunits.
3. The method of claim 1 , wherein said subunits are β subunits.
4. The method of claim 1 , wherein said subunits are βp4 subunits.
5. The method of claim 1 , wherein said subunits are one α subunit and one β subunit.
6. The method of claim 1 , wherein said subunits are co-expressed to form a RSV reverse transcriptase and wherein said RSV reverse transcriptase is isolated from said host cell.
7. The method of claim 1 , wherein said subunits of RSV reverse transcriptase are isolated and then mixed to form a RSV reverse transcriptase.
8. The method of claim 3 , wherein said β subunits form a RSV reverse transcriptase comprising two β subunits.
9. The method of claim 5 , wherein said α and β subunits form a RSV reverse transcriptase comprising an α and a β subunit.
10. The method of claim 1 , wherein said nucleic acid sequences are contained in one or more vectors.
11. The method of claim 1 , wherein said RSV reverse transcriptase has a RNA-dependent DNA polymerase specific activity of 35,000 units per milligram.
12. The method of claim 1 , wherein said RSV reverse transcriptase has a RNA-dependent DNA polymerase specific activity of 30,000 units per milligram to 130,000 units per milligram.
13. The method of claim 1 , wherein said RSV reverse transcriptase has a RNA-dependent DNA polyrnerase specific activity of 30,000 units per milligram to 110,000 units per milligram.
14. The method of claim 1 , wherein said RSV reverse transcriptase has a RNA-dependent DNA polymerase specific activity of 30,000 units per milligram to 90,000 units per milligram.
15. The method of claim 1 , wherein said RSV reverse transcriptase has a RNA-dependent DNA polymerase specific activity of 50,000 units per milligram to 130,000 units per milligram.
16. The method of claim 1 , wherein said RSV reverse transcriptase has a RNA-dependent DNA polyrnerase specific activity of 50,000 units per milligram to 110,000 units per milligram.
17. The method of claim 1 , wherein said RSV reverse transcriptase has a RNA-dependent DNA polymerase specific activity of 50,000 units per milligram to 90,000 units per milligram.
18. The method of claim 1 , wherein said RSV reverse transcriptase has a RNA-dependent DNA polymerase specific activity of 70,000 units per milligram to 130,000 units per milligram.
19. The method of claim 1 , wherein said RSV reverse transcriptase has a RNA-dependent DNA polymerase specific activity of 70,000 units per milligram to 110,000 units per milligram.
20. The method of claim 1 , wherein said RSV reverse transcriptase has a RNA-dependent DNA polymerase specific activity of 70,000 units per milligram 90,000 per milligram.
21. The method of claim 1 , wherein said RSV reverse transcriptase has a RNA-dependent DNA polymerase specific activity from 80,000 units per milligram to 150,000 units per milligram.
22. The method of claim 1 , wherein said RSV reverse transcriptase has a RNA-dependent DNA polyrnerase specific activity of 35,000 units per milligram to 150,000 units per milligram.
23. The method of claim 1 , wherein said RSV reverse transcriptase has a RNA-dependent DNA polymerase specific activity of 40,000 units per milligram to 150,000 units per milligram.
24. The method of claim 1 , wherein said RSV reverse transcriptase has a RNA-dependent DNA polymerase specific activity of 45,000 units per milligram to 150,000 units per milligram.
25. The method of claim 1 , wherein said RSV reverse transcriptase has a RNA-dependent DNA polymerase specific activity of 50,000 units per milligram to 150,000 units per milligram.
26. The method of claim 1 , wherein said RSV reverse transcriptase has a RNA-dependent DNA polymerase specific activity of 55,000 units per milligram to 150,000 units per milligram.
27. The method of claim 1 , wherein said RSV reverse transcriptase has a RNA-dependent DNA polymerase specific activity of 60,000 units per milligram to 150,000 units per milligram.
28. The method of claim 1 , wherein said RSV reverse transcriptase has a RNA-dependent DNA polymerase specific activity of 65,000 units per milligram to 150,000 units per milligram.
29. The method of claim 1 , wherein said RSV reverse transcriptase has a RNA-dependent DNA polymerase specific activity of 70,000 units per milligram to 150,000 units per milligram.
30. The method of claim 1 , wherein said RSV reverse transcriptase has a RNA-dependent DNA polymerase specific activity of 75,000 units per milligram to 150,000 units per milligram.
31. The method of claim 10 , wherein said nucleic acid sequences are contained on the same vector.
32. The method of claim 10 , wherein said nucleic acid sequences are contained on different vectors.
33. The method of claim 1 , wherein said host cell is a cultured insect cell.
34. The method of claim 1 , wherein said host cell is an insect larva cell.
35. The method of claim 1 , wherein said host cell is a yeast cell.
36. A method of producing a Rous Sarcoma Virus (RSV) reverse transcriptase having a RNA-dependent DNA polymerase specific activity of 30,000 units per milligram to 150,000 units per milligram or a mutant RSV reverse transcriptase having reduced RNase H activity, said method comprising
(a) obtaining a eukaryotic host cell comprising one or more nucleic acid sequences encoding one or more mutant subunits of RSV reverse transcriptase having reduced RNase H activity relative to the wild-type reverse transcriptase;
(b) culturing said host cell under conditions sufficient to produce said mutant RSV reverse transcriptase; and
(c) isolating or purifying said reverse trascriptase thereby obtaining a mutant RSV reverse transcriptase having a RNA-dependent DNA polymerase specific activity of 30,000 units per milligram to 150,000 units per milligram.
37. The method of claim 36 , wherein said one or more nucleic acid sequences encoding one or more subunits of RSV reverse transcriptase are contained in one or more vectors subunits of said RSV reverse transcriptase are expressed in said host cell to form said mutant RSV reverse trascriptase.
38. The method of claim 36 , wherein said mutant RSV reverse transcriptase subunits are selected from the group consisting of α subunits, β subunits and βp4 subunits.
39. The method of claim 38 , wherein said mutant subunits are a subunits.
40. The method of claim 38 , wherein said mutant subunits are β subunits.
41. The method of claim 38 , wherein said mutant subunits are βp4 subunits.
42. The method of claim 38 , wherein said mutant subunits are one α subunit and one β subunit.
43. The method of claim 36 , wherein said subunits are co-expressed to form a RSV reverse transcriptase and wherein said RSV reverse transcriptase is isolated from said host cell.
44. The method of claim 36 , wherein said subunits of RSV reverse transcriptase are isolated and then mixed to form a RSV reverse transcriptase.
45. The method of claim 40 , wherein said mutant β subunits form a RSV reverse transcriptase comprising two β subunits.
46. The method of claim 42 , wherein said mutant a and β subunits form a RSV reverse transcriptase comprising an α and a β subunit.
47. The method of claim 36 , wherein said subunits are encoded by one or more nucleotides sequences contained on the same or on different vectors nucleic acid sequences are contained in one or more vectors.
48. The method of claim 36 , wherein said mutant RSV reverse transcriptase has a RNA-dependent DNA polymerase specific activity of 35,000 units per milligram to 180,000 units per milligram.
49. The method of claim 36 , wherein said mutant RSV reverse transcriptase has a RNA-dependent DNA polymerase specific activity of 30,000 units per milligram to 110,000 units per milligram.
50. The meted of claim 36 , wherein said mutant RSV reverse transcriptase has a RNA-dependent DNA polymerase specific activity of 30,000 units per milligram to 110,000 units per milligram.
51. The method of claim 36 , wherein said mutant RSV reverse transcriptase has a RNA-dependent DNA polymerase specific activity of 30,000 units per milligram to 90,000 units per milligram.
52. The method of claim 36 , wherein said mutant RSV reverse transcriptase has a RNA-dependent DNA polymerase specific activity of 50,000 units per milligram to 130,000 units per milligram.
53. The method of claim 36 , wherein said mutant RSV reverse transcriptase has a RNA-dependent DNA polymerase specific activity of 50,000 units per milligram to 110,000 units per milligram.
54. The method of claim 36 , wherein said mutant RSV reverse transcriptase has a RNA-dependent DNA polymerase specific activity of 50,000 units per milligram to 90,000 units per milligram.
55. The method of claim 36 , wherein said mutant RSV reverse transcriptase has a RNA-dependent DNA polymerase specific activity of 70,000 units per milligram to 130,000 units per milligram.
56. The method of claim 36 , wherein said mutant RSV reverse transcriptase has a RNA-dependent DNA polymerase specific activity of 70,000 units per milligram to 110,000 unit per milligram.
57. The method of claim 36 , wherein said mutant RSV reverse franscriptase has a RNA-dependent DNA polymerase specific activity of 70,000 units per milligram to 90,000 units per milligram.
58. The method of claim 47 , wherein said nucleic acid sequences are contained on the same vector.
59. The method of claim 47 , wherein said nucleic acid sequences are contained on different vectors.
60. The method of claim 36 , wherein said host cell is a cultured insect cell.
61. The method of claim 36 , wherein said host cell is an insect larva cell.
62. The method of claim 36 , wherein said host cell is a yeast cell.