Chimaeric gene coding for a transit peptide and a heterologous peptide

• US 6,063,601 A
• Filed: 06/06/1995
• Issued: 05/16/2000
• Est. Priority Date: 12/28/1984
• Status: Expired due to Term

• Current Assignee: Bayer Cropscience NV (Bayer AG)
• Original Assignee: Bayer AG, Plant Genetic Systems NV (Bayer AG)
• Inventors: Schreier, Peter, Kausch, Albert P., Schell, Jeff, Timko, Michael P., Herrera-Estrella, Luis, Van Den Broeck, Guidi, Cash more, Anthony R., Bohnert, Hans J., Van Montagu, Marc
• Primary Examiner(s): Fox, David T.
• Application Number:
  US08/468,317
• Time in Patent Office: 1,806 Days
• Field of Search: 435/69.7, 435/69.8, 435/70.1, 435/172.3, 435/419, 536/23.4, 536/24.1, 536/23.6, 536/23.7, 800/205
• US Class Current: 800/278
• CPC Class Codes: C12N 15/8216 Methods for controlling, regulating or enhancing expression of transgenes in plant cells
• CPC Class Codes: C12N 15/8221 Transit peptides

Abstract

Chimaeric DNA sequence which encodes: 1) a transit peptide of a cytoplasmic precursor of a chloroplast protein or polypeptide of a plant and 2) a protein or polypeptide that is heterologous to the transit peptide. The chimaeric DNA sequence can be used as a vector for transforming a plant cell so that a chimaeric precursor of the heterologous protein or polypeptide is produced in the cytoplasm of the cell and the chimaeric precursor then transports the heterologous protein or polypeptide in vivo into a chloroplast of the cell.

Claims

1. A process for achieving expression of a protein or polypeptide of interest in cells of a plant, said protein or polypeptide of interest being different from a mature chloroplast protein or chloroplast polypeptide derived from a natural cytoplasmic precursor thereof, said process comprising the step of:

(1) introducing into the nuclear genome of the said plant a chimaeric DNA sequence comprising;

a) a nucleic acid sequence coding for a chimaeric protein or polypeptide comprising, in sequence, a transit peptide of a cytoplasmic precursor of a chloroplast protein or chloroplast polypeptide of a plant species, and said protein or polypeptide of interest which is different from said mature chloroplast protein or chloroplast polypeptide,

b) a promoter upstream of said nucleic acid sequence recognized by polymerases endogenous to said plant for control of transcription of said nucleic acid sequence in said cells;

wherein said nucleic acid sequence is expressed in cells of said plant under control of said promoter, and the protein or polypeptide of interest is translocated into chloroplasts of cells of said plant.

2. The process of claim 1 in which said protein or polypeptide of interest is of bacterial origin.

3. The process of claim 1 in which said protein or polypeptide of interest is of plant origin.

4. The process of claim 1 in which said protein or polypeptide of interest confers resistance to an herbicide.

5. The process of claim 1 in which the first amino acid of said protein or polypeptide of interest is a methionine.

6. The process of claim 1 in which no more than the first seven amino acids of said protein or polypeptide of interest are encoded by a synthetic nucleotide linker.

7. The process of claim 1 in which the transit peptide is from a cytoplasmic precursor of a chloroplast protein which is selected from the group consisting of a small subunit of ribulose-1,5-bisphosphate carboxylase and chlorophyll a/b binding proteins.

8. The process of claim 7 in which the transit peptide is from a cytoplasmic precursor of the small subunit of ribulose-1,5-bisphosphate carboxylase of a plant selected from the group consisting of soybean, pea, duckweed and wheat.

9. The process of claim 7 in which the transit peptide comprises a sequences:

M-A-S-M-I-S-S-S-A-V-T-T-V-S-R-A-S-R-G-Q-S-A-A-V-A-P-F-G-G-L-K-S-M-T-G-F-P-V-K-K-V-N-T-D-I-T-S-I-T-S-N-G-G-R-V-K-C;

or a sequence;

M-A-A-S-S-S-S-S-M-A-L-S-S-P-T-L-A-G-K-Q-L-K-L-N-P-S-S-Q-E-I-G-A-A-R-P-T.

10. The process of claim 1 in which said promoter is normally associated with the DNA encoding said transit peptide.

11. The process of claim 10 in which said promoter is a promoter of a plastocyanine gene, a ferredoxin-NADP+ oxydoreductase gene, a ribulose-1,5-bisphosphate carboxylase gene, or a chlorophyll a/b binding protein gene.

12. The process of claim 1 in which said promoter is foreign to the DNA encoding said transit peptide.

13. The process of claim 12 in which said promoter is a promoter of a nopaline synthase gene.

41. A process to produce a seed capable of growing into a plant comprising a protein or polypeptide of interest which is translocated into chloroplasts of cells of said plant, said process comprising the step of producing a plant of claim 1 and harvesting from said plant a seed comprising said chimaeric DNA sequence.

14. A process to produce a plant comprising a protein of polypeptide of interest which is part of a fusion protein and which is translocated into chloroplasts of cells of said plant, said process comprising the step of introducing into the nuclear genome of said plant a chimaeric DNA sequence comprising:

a) a nucleic acid sequence coding for a chimaeric protein or polypeptide comprising in sequence;

1) a transit peptide included in a cytoplasmic precursor of a chloroplast protein or chloroplast polypeptide of a plant species, and,

2) a fusion protein comprising in sequence;

2.1) the N-terminal part of a mature chloroplast protein or chloroplast polypeptide derived from a natural cytoplasmic precursor thereof, and,

2.2) a protein or polypeptide of interest which is different from said mature chloroplast protein or chloroplast polypeptide and,

b) a promoter upstream of said nucleic acid sequence recognized by polymerases endogenous to said plant, wherein the chimaeric DNA sequence is expressed in cells of said plant under control of said promoter.

15. The process of claim 14 in which said protein or polypeptide of interest is of bacterial origin.

16. The process of claim 14 in which said protein or polypeptide of interest is of plant origin.

17. The process of claim 14 in which said protein or polypeptide of interest confers resistance to an herbicide.

18. The process of claim 14 in which said N-terminal part is encoded by a DNA sequence that contains an intron.

19. The process of claim 14 in which the transit peptide is from a cytoplasmic precursor of a chloroplast protein which is selected from the group consisting of a small subunit of ribulose-1,5-bisphosphate carboxylase and chlorophyll a/b binding proteins.

20. The process of claim 19 in which the transit peptide is from a cytoplasmic precursor of the small subunit of ribulose-1,5-bisphosphate carboxylase of a plant selected from the group consisting of soybean, pea, duckweed and wheat.

21. The process of claim 20 in which the transit peptide comprises a sequence:

M-A-S-M-I-S-S-S-A-V-T-T-V-S-R-A-S-R-G-Q-S-A-A-V-A-P-F-G-G-L-K-S-M-T-G-F-P-V-K-K-V-N-T-D-I-T-S-I-T-S-N-G-G-R-V-K-C, or

M-A-A-S-S-S-S-S-M-A-L-S-S-P-T-L-A-G-K-Q-L-K-L-N-P-S-S-Q-E-I-G-A-A-R-P-T.

22. The process of claim 14 in which said promoter is normally associated with the DNA encoding said transit peptide.

23. The process of claim 22 in which said promoter is a promoter of a gene selected from the group consisting of a plastocyanine gene, a ferredoxin-NADP+ oxydoreductase gene, a ribulose-1,5-bisphosphate carboxylase gene, and a chlorophyll a/b binding protein gene.

24. The process of claim 14 in which said promoter is foreign to the DNA encoding said transit peptide.

25. The process of claim 24 in which said promoter is a promoter of a nopaline synthase gene.

42. A process to produce a seed capable of growing into a plant comprising a protein or polypeptide of interest which is including in a fusion protein which is translocated into chloroplasts of cells of said plant, said process comprising the step of producing a plant of claim 14 and harvesting from said plant a seed comprising said chimaeric DNA sequence.

26. A process to produce a plant comprising a protein or polypeptide of interest which is part of a fusion protein and which is translocated into chloroplasts of cells of said plant, said process comprising the step of introducing into the nuclear genome of said plant a chimaeric DNA sequence comprising:

a) a nucleic acid sequence coding for a chimaeric protein or polypeptide comprising in sequence;

1) a transit peptide included in a cytoplasmic precursor of a chloroplast protein or chloroplast polypeptide of a plant species, and,

2) a fusion protein comprising in sequence;

2.1) a N-terminal part including no more than the first 22 N-terminal amino acids of a mature chloroplast protein or chloroplast polypeptide derived from a natural cytoplasmic precursor thereof, and

2.2) a protein or polypeptide of interest which is different from said mature chloroplast protein or chloroplast polypeptide and,

b) a promoter upstream of said nucleic acid sequence recognized by polymerases endogenous to said plant, wherein the chimaeric DNA sequence is expressed in cells of said plant under control of said promoter.

27. The process of claim 26 in which said protein or polypeptide of interest is of bacterial origin.

28. The process of claim 26 in which said protein or polypeptide of interest is of plant origin.

29. The process of claim 26 in which said protein or polypeptide of interest confers resistance to an herbicide.

30. The process of claim 26 in which said N-terminal part is encoded by a DNA sequence that includes an intron.

31. The process of claim 26 in which said N-terminal part is from a small subunit of a ribulose-1,5-bisphosphate carboxylase of Pisum sativum.

32. The process of claim 26 in which said N-terminal part comprises no more than the first five N-terminal amino acids of said mature chloroplast protein or polypeptide.

33. The process of claim 32 in which said N-terminal part comprises the sequence M-Q-V-W-P.

34. The process of claim 26 in which the transit peptide is from a cytoplasmic precursor of a chloroplast protein which is selected from the group consisting of a small subunit of ribulose-1,5-biphosphate carboxylase and chlorophyll a/b binding proteins.

35. The process of claim 34 in which the transit peptide is from a cytoplasmic precursor of the small subunit of ribulose-1,5-bisphosphate carboxylase of a plant selected from the group consisting of soybean, pea, duckweed and wheat.

36. The process of claim 35 in which the transit peptide comprises a sequence:

M-A-S-M-I-S-S-S-A-V-T-T-V-S-R-A-S-R-G-Q-S-A-A-V-A-P-F-G-G-L-K-S-M-T-G-F-P-V-K-K-V-N-T-D-I-T-S-I-T-S-N-G-G-R-V-K-C, or

M-A-A-S-S-S-S-S-M-A-L-S-S-P-T-L-A-G-K-Q-L-K-L-N-P-S-S-Q-E-I-G-A-A-R-P-T.

37. The process of claim 26 in which said promoter is normally associated with the DNA encoding said transit peptide.

38. The process of claim 37 in which the promoter is a promoter of a gene selected from the group consisting of a plastocyanine gene, a ferredoxin-NADP+ oxydoreductase gene, a ribulose-1,5-bisphosphate gene, and a chlorophyll a/b binding protein gene.

39. The process of claim 26 in which said promoter is foreign to the DNA encoding said transit peptide.

40. The process of claim 39 in which said promoter is a promoter of a nopaline synthase gene.

43. A process to produce a seed capable of growing into a plant comprising a protein or polypeptide of interest which is included in a fusion protein which is translocated into chloroplasts of cells of said plant, said process comprising the step of producing a plant of claim 26 and harvesting from said plant a seed comprising said chimaeric DNA sequence.

44. A process for achieving expression of a protein or polypeptide of interest in cells of a plant, said protein or polypeptide of interest being different from a mature chloroplast protein or chloroplast polypeptide derived from a natural cytoplasmic precursor thereof, said process comprising the step of:

producing a plant comprising in its nuclear genome a chimaeric DNA sequence comprising;

a) a nucleic acid sequence coding for a chimaeric protein or polypeptide comprising, in sequence, a transit peptide of a cytoplasmic precursor of a chloroplast protein or chloroplast polypeptide of a plant species, and said protein or polypeptide of interest which is different from said mature chloroplast protein or chloroplast polypeptide,

b) a promoter upstream of said nucleic acid sequence for control of transcription of said nucleic acid sequence in said cells,

wherein said nucleic acid sequence is expressed in cells of said plant under control of said promoter and the protein of polypeptide of interest is translocated into chloroplasts of cells of said plant.

45. The process of claim 44 in which said protein or polypeptide of interest is of bacterial origin.

46. The process of claim 44 in which said protein or polypeptide of interest is of plant origin.

47. The process of claim 44 in which said protein or polypeptide of interest confers resistance to an herbicide.

48. The process of claim 44 in which the first amino acid of said protein or polypeptide of interest is a methionine.

49. The process of claim 44 in which no more than the first seven amino acids of said protein or polypeptide of interest are encoded by a synthetic nucleotide linker.

50. The process of claim 44 in which the transit peptide is from a cytoplasmic precursor of a chloroplast protein which is selected from the group consisting of a small subunit of ribulose-1,5-biphosphate carboxylase and chlorophyll a/b binding proteins.

51. The process of claim 50 in which the transit peptide is from a cytoplasmic precursor of the small subunit of ribulose-1,5-biphosphate carboxylase of a plant selected from the group consisting of soybean, pea, duckweed and wheat.

52. The process of claim 50 in which the transit peptide comprises a sequence:

M-A-S-M-I-S-S-S-A-V-T-T-V-S-R-A-S-R-G-Q-S-A-A-V-A-P-F-G-G-L-K-S-M-T-G-F-P-V-K-K-V-N-T-D-I-T-S-I-T-S-N-G-G-R-V-K-C or a sequence;

M-A-A-S-S-S-S-S-M-A-L-S-S-P-T-L-A-G-K-Q-L-K-L-N-P-S-S-Q-E-I-A-A-R-P-T.

53. The process of claim 44 in which said promoter is normally associated with the DNA encoding said transit peptide.

54. The process of claim 53 in which said promoter is a promoter of a plastocyanine gene, a ferredoxin-NADP+ oxydoreductase gene, a ribulose-1,5-biphosphate carboxylase gene or a chlorophyll a/b binding protein gene.

55. The process of claim 44 in which said promoter is foreign to the DNA encoding said transit peptide.

56. The process of claim 55 in which said promoter is a promoter of a nopaline synthase gene.

85. A process to produce a seed capable of growing into a plant comprising a protein or polypeptide of interest which is translocated into chloroplasts of cells of said plant, said process comprising the step of producing a plant of claim 44 and harvesting from said plant a seed comprising said chimaeric DNA sequence.

57. A process to produce a plant comprising a protein or polypeptide of interest which is part of a fusion protein and which is translocated into chloroplasts of cells of said plant, said process comprising the step of producing a plant comprising in its nuclear genome a chimeric DNA sequence comprising:

a) a nucleic acid sequence coding for a chimaeric protein or polypeptide comprising in sequence;

1) a transit peptide included in a cytoplasmic precursor of a chloroplast protein or chloroplast polypeptide of a plant species, and,

2) a fusion protein comprising in sequence;

2.1) the N-terminal part of a mature chloroplast protein or chloroplast polypeptide derived from a natural cytoplasmic precursor thereof, and,

2.2) a protein or polypeptide of interest which is different from said mature chloroplast protein or chloroplast polypeptide and,

b) a promoter upstream of said nucleic acid sequence recognized by polymerases endogenous to said plant, wherein the chimaeric DNA sequence is expressed in cells of said plant under control of said promoter.

58. The process of claim 57 in which said protein or polypeptide of interest is of bacterial origin.

59. The process of claim 57 in which said protein or polypeptide of interest is of plant origin.

60. The process of claim 57 in which said protein or polypeptide of interest confers resistance to an herbicide.

61. The process of claim 57 in which said N-terminal part is encoded by a DNA sequence that contains an intron.

62. The process of claim 57 in which the transit peptide is from a cytoplasmic precursor of a chloroplast protein which is selected from the group consisting of a small subunit of ribulose-1,5-biphosphate carboxylase and chlorophyll a/b binding proteins.

63. The process of claim 62 in which the transit peptide is from a cytoplasmic precursor of the small subunit of ribulose-1,5-biphosphate carboxylase of a plant selected from the group consisting of soybean, pea, duckweed and wheat.

64. The process of claim 63 in which the transit peptide comprising a sequence:

M-A-S-M-I-S-S-S-A-V-T-T-V-S-R-A-S-R-G-Q-S-A-A-V-A-P-F-G-G-L-K-S-M-T-G-F-P-V-K-K-V-N-T-D-I-T-S-I-T-S-N-G-G-R-V-K-C or a sequence;

M-A-A-S-S-S-S-S-M-A-L-S-S-P-T-L-A-G-K-Q-L-K-L-N-P-S-S-Q-E-I-G-A-A-R-P-T.

65. The process of claim 57 in which said promoter is normally associated with the DNA encoding said transit peptide.

66. The process of claim 65 in which said promoter is a promoter of a plastocyanine gene, a ferredoxin-NADP+ oxydoreductase gene, a ribulose-1,5-biphosphate carboxylase gene or a chlorophyll a/b binding protein gene.

67. The process of claim 57 in which said promoter is foreign to the DNA encoding said transit peptide.

68. The process of claim 67 in which said promoter is a promoter of a nopaline synthase gene.

86. A process to produce a seed capable of growing into a plant comprising a protein or polypeptide of interest which is translocated into chloroplasts of cells of said plant, said process comprising the step of producing a plant of claim 57 and harvesting from said plant a seed comprising said chimaeric DNA sequence.

69. A process to produce a plant comprising a protein or polypeptide of interest which is part of a fusion protein and which is translocated into chloroplasts of cells of said plant, said process comprising the step of producing a plant comprising in its nuclear genome a chimaeric DNA sequence comprising:

a) a nucleic acid sequence coding for a chimaeric protein or polypeptide comprising in sequence;

1) a transit peptide included in a cytoplasmic precursor of a chloroplast protein or chloroplast polypeptide of a plant species, and,

2) a fusion protein comprising in sequence;

2.1) a N-terminal part including no more than the first 22 N-terminal amino acids of a mature chloroplast protein or chloroplast polypeptide derived from a natural cytoplasmic precursor thereof, and

71. The process of claim 69 in which said protein or polypeptide of interest is of bacterial origin.

72. The process of claim 69 in which said protein or polypeptide of interest is of plant origin.

73. The process of claim 69 in which said protein or polypeptide of interest confers resistance to an herbicide.

74. The process of claim 69 in which said N-terminal part is encoded by a DNA sequence that includes an intron.

75. The process of claim 69 in which said N-terminal part is from a small subunit of ribulose-1,5-biphosphate carboxylase of Pisum sativum.

76. The process of claim 69 in which said N-terminal part comprises no more than the first five N-terminal amino acids of said mature chloroplast protein or chloroplast polypeptide.

77. The process of claim 75 in which said N-terminal part comprises the sequence M-Q-V-W-P.

78. The process of claim 69 in which the transit peptide is from a cytoplasmic precursor of a chloroplast protein which is selected from the group consisting of a small subunit of ribulose-1,5-biphosphate carboxylase and chlorophyll a/b binding proteins.

79. The process of claim 77 in which the transit peptide is from a cytoplasmic precursor of the small subunit of ribulose-1,5-biphosphate carboxylase of a plant selected from the group consisting of soybean, pea, duckweed and wheat.

80. The process of claim 78 in which the transit peptide comprises a sequence:

M-A-S-M-I-S-S-S-A-V-T-T-V-S-R-A-S-R-G-Q-S-A-A-V-A-P-F-G-G-L-K-S-M-T-G-F-P-V-K-K-V-N-T-D-I-T-S-I-T-S-N-G-G-R-V-K-C or a sequence;

M-A-A-S-S-S-S-S-M-A-L-S-S-P-T-L-A-G-K-Q-L-K-L-N-P-S-S-Q-E-I-G-A-A-R-P-T.

81. The process of claim 69 in which said promoter is normally associated with the DNA encoding said transmit peptide.

82. The process of claim 80 in which said promoter is a promoter of a plastocyanine gene, a ferredoxin-NADP+ oxydoreductase gene, a ribulose-1,5-biphosphate carboxylase gene or a chlorophyll a/b binding protein gene.

83. The process of claim 69 in which said promoter is foreign to the DNA encoding said transit peptide.

84. The process of claim 82 in which said promoter is a promoter of a nopaline synthase gene.

87. A process to produce a seed capable of growing into a plant comprising a protein or polypeptide of interest which is translocated into chloroplasts of cells of said plant, said process comprising the step of producing a plant of claim 69 and harvesting from said plant a seed comprising said chimaeric DNA sequence.

70. 2) a protein or polypeptide of interest which is different from said mature chloroplast protein or chloroplast polypeptide and,

b) a promoter upstream of said nucleic acid sequence recognized by polymerases endogenous to said plant, wherein the chimaeric DNA sequence is expressed in cells of said plant under control of said promoter.