Humanized green fluorescent protein genes and methods

• US 5,874,304 A
• Filed: 01/18/1996
• Issued: 02/23/1999
• Est. Priority Date: 01/18/1996
• Status: Expired due to Term

• Current Assignee: University of Florida Research Foundation, Incorporated (State University System of Florida)
• Original Assignee: University of Florida Research Foundation, Incorporated (State University System of Florida)
• Inventors: Hauswirth, William W., Muzyczka, Nicholas, Zolotukhin, Sergei
• Primary Examiner(s): Degen, Nancy
• Assistant Examiner(s): WANG, ANDREW J
• Application Number:
  US08/588,201
• Time in Patent Office: 1,132 Days
• Field of Search: 435/6, 435/172.3, 435/173.3, 435/235.1, 435/325, 435/320.1, 536/23.1
• US Class Current: 435/366
• CPC Class Codes: C07K 14/43595 from coelenteratae, e.g. medusae
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C12N 15/87 Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
• CPC Class Codes: C12N 2799/025 where the vector is derived from a parvovirus
• CPC Class Codes: C12N 2799/027 where the vector is derived from a retrovirus

Abstract

Disclosed are synthetic and "humanized" versions of green fluorescent protein (GFP) genes adapted for high level expression in mammalian cells, especially those of human origin. Base substitutions are made in various codons in order to change the codon usage to one more appropriate for expression in mammalian cells. Recombinant vectors carrying such humanized genes are also disclosed. In addition, various methods for using the efficient expression of humanized GFP in mammalian cells and in animals are described.

Claims

1. A humanized green fluorescent protein (GFP) gene.

2. The humanized GFP gene of claim 1, wherein said gene encodes a green fluorescent protein that has the amino acid sequence of SEQ ID NO:

2.

3. The humanized GFP gene of claim 1, wherein said gene encodes a green fluorescent protein that has the amino acid sequence of SEQ ID NO:

2 in which Serine at position 65 has been replaced by Threonine.

4. The humanized GFP gene of claim 1, wherein said gene encodes a green fluorescent protein that has the amino acid sequence of SEQ ID NO:

2 in which Tyrosine at position 66 has been replaced by Histidine.

5. The humanized GFP gene of claim 1, wherein said gene encodes a green fluorescent protein that has the amino acid sequence of SEQ ID NO:

2 in which the chromophore sequence Phe Ser Tyr Gly Val Gln (SEQ ID NO;

4) between positions 64 and 69 has been replaced by the sequence Met Gly Tyr Gly Val Leu (SEQ ID NO;

5).

6. The humanized GFP gene of claim 1, wherein at least about 10% of said codon positions contain a humanized codon.

7. The humanized GFP gene of claim 6, wherein at least about 15% of said codon positions contain a humanized codon.

8. The humanized GFP gene of claim 7, wherein at least about 20% of said codon positions contain a humanized codon.

9. The humanized GFP gene of claim 8, wherein at least about 25% of said codon positions contain a humanized codon.

10. The humanized GFP gene of claim 9, wherein at least about 30% of said codon positions contain a humanized codon.

11. The humanized GFP gene of claim 10, wherein at least about 35% of said codon positions contain a humanized codon.

12. The humanized GFP gene of claim 11, wherein at least about 50% of said codon positions contain a humanized codon.

13. The humanized GFP gene of claim 1, wherein said gene comprises at least seven humanized codons from the 10 codons located at codon positions 18, 53, 93, 125, 150, 178, 195, 208, 236 and 224 of the GFP gene sequence.

14. The humanized GFP gene of claim 13, wherein said gene comprises at least eight humanized codons from the 10 codons located at codon positions 18, 53, 93, 125, 150, 178, 195, 208, 236 and 224 of the GFP gene sequence.

15. The humanized GFP gene of claim 14, wherein said gene comprises at least nine humanized codons from the 10 codons located at codon positions 18, 53, 93, 125, 150, 178, 195, 208, 236 and 224 of the GFP gene sequence.

16. The humanized GFP gene of claim 15, wherein said gene comprises a humanized codon at each of the codon positions 18, 53, 93, 125, 150, 178, 195, 208, 236 and 224 of the GFP gene sequence.

17. The humanized GFP gene of claim 13, wherein said gene comprises any one of the humanized Leucine codons CTG, CTC or TTG at codon positions 18, 53, 125, 178, 195 and 236 of the GFP gene sequence.

18. The humanized GFP gene of claim 13, wherein said gene comprises the humanized Valine codon GTG at codon positions 93, 150 and 224 of the GFP gene sequence.

19. The humanized GFP gene of claim 13, wherein said gene comprises the humanized Serine codon TCT at codon position 208 of the GFP gene sequence.

20. The humanized GFP gene of claim 1, wherein said gene comprises an increased number of GCC or GCT Alanine-encoding codons within the coding region in comparison to the wild type jellyfish gene sequence of SEQ ID NO:

1.

21. The humanized GFP gene of claim 1, wherein said gene comprises an increased number of TGC Cysteine-encoding codons within the coding region in comparison to the wild type jellyfish gene sequence of SEQ ID NO:

1.

22. The humanized GFP gene of claim 1, wherein said gene comprises an increased number of GAC Aspartate-encoding codons within the coding region in comparison to the wild type jellyfish gene sequence of SEQ ID NO:

1.

23. The humanized GFP gene of claim 1, wherein said gene comprises an increased number of GAG Glutamate-encoding codons within the coding region in comparison to the wild type jellyfish gene sequence of SEQ ID NO:

1.

24. The humanized GFP gene of claim 1, wherein said gene comprises an increased number of TTC Phenylalanine-encoding codons within the coding region in comparison to the wild type jellyfish gene sequence of SEQ ID NO:

1.

25. The humanized GFP gene of claim 1, wherein said gene comprises an increased number of GGC Glycine-encoding codons within the coding region in comparison to the wild type jellyfish gene sequence of SEQ ID NO:

1.

26. The humanized GFP gene of claim 1, wherein said gene comprises an increased number of CAC Histidine-encoding codons within the coding region in comparison to the wild type jellyfish gene sequence of SEQ ID NO:

1.

27. The humanized GFP gene of claim 1, wherein said gene comprises an increased number of ATC Isoleucine-encoding codons within the coding region in comparison to the wild type jellyfish gene sequence of SEQ ID NO:

1.

28. The humanized GFP gene of claim 1, wherein said gene comprises an increased number of AAG Lysine-encoding codons within the coding region in comparison to the wild type jellyfish gene sequence of SEQ ID NO:

1.

29. The humanized GFP gene of claim 1, wherein said gene comprises an increased number of CTG or CTC Leucine-encoding codons within the coding region in comparison to the wild type jellyfish gene sequence of SEQ ID NO:

1.

30. The humanized GFP gene of claim 1, wherein said gene comprises an increased number of AAC Asparagine-encoding codons within the coding region in comparison to the wild type jellyfish gene sequence of SEQ ID NO:

1.

31. The humanized GFP gene of claim 1, wherein said gene comprises an increased number of CCC or CCT Proline-encoding codons within the coding region in comparison to the wild type jellyfish gene sequence of SEQ ID NO:

1.

32. The humanized GFP gene of claim 1, wherein said gene comprises an increased number of CAG Glutamine-encoding codons within the coding region in comparison to the wild type jellyfish gene sequence of SEQ ID NO:

1.

33. The humanized GFP gene of claim 1, wherein said gene comprises an increased number of CGC, AGG or CGG Arginine-encoding codons within the coding region in comparison to the wild type jellyfish gene sequence of SEQ ID NO:

1.

34. The humanized GFP gene of claim 1, wherein said gene comprises an increased number of AGC or TCC Serine-encoding codons within the coding region in comparison to the wild type jellyfish gene sequence of SEQ ID NO:

1.

35. The humanized GFP gene of claim 1, wherein said gene comprises an increased number of ACC Threonine-encoding codons within the coding region in comparison to the wild type jellyfish gene sequence of SEQ ID NO:

1.

36. The humanized GFP gene of claim 1, wherein said gene comprises an increased number of GTG or GTC Valine-encoding codons within the coding region in comparison to the wild type jellyfish gene sequence of SEQ ID NO:

1.

37. The humanized GFP gene of claim 1, wherein said gene comprises an increased number of TAC Tyrosine-encoding codons within the coding region in comparison to the wild type jellyfish gene sequence of SEQ ID NO:

1.

38. The humanized GFP gene of claim 1, wherein said gene comprises a TGA termination codon.

39. The humanized GFP gene of claim 1, wherein said gene comprises a decreased number of GCA Alanine-encoding codons within the coding region in comparison to the wild type jellyfish gene sequence of SEQ ID NO:

1.

40. The humanized GFP gene of claim 1, wherein said gene comprises a decreased number of GGU Glycine-encoding codons within the coding region in comparison to the wild type jellyfish gene sequence of SEQ ID NO:

1.

41. The humanized GFP gene of claim 1, wherein said gene comprises a decreased number of CTT, CTA or TTA Leucine-encoding codons within the coding region in comparison to the wild type jellyfish gene sequence of SEQ ID NO:

1.

42. The humanized GFP gene of claim 1, wherein said gene comprises a decreased number of AGA Arginine-encoding codons within the coding region in comparison to the wild type jellyfish gene sequence of SEQ ID NO:

1.

43. The humanized GFP gene of claim 1, wherein said gene comprises a decreased number of AGT, TCA or TCG Serine-encoding codons within the coding region in comparison to the wild type jellyfish gene sequence of SEQ ID NO:

1.

44. The humanized GFP gene of claim 1, wherein said gene comprises a decreased number of GTT or GTA Valine-encoding codons within the coding region in comparison to the wild type jellyfish gene sequence of SEQ ID NO:

1.

45. The humanized GFP gene of claim 1, wherein said gene is operatively positioned downstream from a Kozak consensus sequence.

46. The humanized GFP gene of claim 1, wherein said gene comprises the nucleic acid sequence of SEQ ID NO:

3.

47. The humanized GFP gene of claim 1, wherein said gene is operatively linked to protein-encoding nucleic acid sequence.

48. The humanized GFP gene of claim 1, wherein said gene is positioned under the transcriptional control of a promoter operative in a mammalian cell.

49. The humanized GFP gene of claim 48, further defined as a recombinant vector.

50. An expression vector comprising a humanized GFP reporter gene operatively positioned downstream from a promoter, the promoter directing expression of the humanized GFP gene in a mammalian cell.

51. The expression vector of claim 50, wherein said promoter is a constitutive promoter.

52. The expression vector of claim 50, wherein said promoter is a viral promoter.

53. The expression vector of claim 50, wherein said promoter is a HSV, TK, RSV, SV40, CMV or β

-actin promoter.

54. The expression vector of claim 53, wherein said promoter is a CMV promoter.

55. The expression vector of claim 50, wherein said promoter is an inducible promoter.

56. The expression vector of claim 55, wherein said promoter is a cytochrome P450, heat shock protein, metallothionein or estrogen gene promoter, a radiation-inducible promoter or a tetVP16 promoter.

57. The expression vector of claim 50, wherein said promoter is a tissue-specific promoter.

58. The expression vector of claim 57, wherein said promoter is a FAB, insulin, transphyretin, α

1-antitrypsin, PAI-1, apolipoprotein AI, LDL receptor, MBP, GFAP, OPSIN or NSE gene promoter.

59. The expression vector of claim 50, wherein said expression vector further comprises a multiple cloning site.

60. The expression vector of claim 59, wherein said expression vector comprises a multiple cloning site operatively positioned between said promoter and said humanized GFP gene.

61. The expression vector of claim 59, wherein said expression vector comprises a multiple cloning site operatively positioned downstream from said humanized GFP gene.

62. The expression vector of claim 50, wherein said expression vector further comprises an IRES element.

63. The expression vector of claim 50, wherein said expression vector further comprises a second reporter gene.

64. The expression vector of claim 63, wherein said second reporter gene is comprised within a second transcriptional unit.

65. The expression vector of claim 63, wherein said second reporter gene confers resistance to neomycin, hygromycin, puromycin, zeocin, mycophenolic acid, histidinol or methotrexate.

66. The expression vector of claim 50, wherein said expression vector further comprises a polyadenylation signal.

67. The expression vector of claim 50, wherein said expression vector is a recombinant adenoviral vector.

68. The expression vector of claim 50, wherein said expression vector is a recombinant adeno-associated viral (AAV) vector.

69. The expression vector of claim 50, wherein said expression vector is a recombinant retroviral vector.

70. The expression vector of claim 50, wherein said expression vector comprises a humanized GFP reporter gene that has the nucleic acid sequence of SEQ ID NO:

3.

71. A recombinant host cell comprising a humanized GFP gene.

72. The recombinant host cell of claim 71, wherein said humanized GFP gene is introduced into said cell by means of a recombinant vector.

73. The recombinant host cell of claim 72, wherein said cell expresses said humanized GFP gene to produce the encoded GFP protein.

74. The recombinant host cell of claim 71, wherein said cell is a mammalian cell.

75. The recombinant host cell of claim 71, wherein said cell is a human cell.

76. The recombinant host cell of claim 72, wherein said cell is a VERO, HeLa, CHO, COS, W138, BHK, HepG2, 3T3, RIN, MDCK, A549, PC12, K562 or 293 cell.

77. The recombinant host cell of claim 71, wherein said cell is a cell of a primary cell line.

78. The recombinant host cell of claim 71, wherein said cell is located within a mammal.

79. The recombinant host cell of claim 71, wherein said cell comprises a humanized GFP gene that comprises the nucleic acid sequence of SEQ ID NO:

3.

80. The recombinant host cell of claim 71, wherein said cell further comprises a recombinant gene that expresses a desired protein.

81. A reporter gene expression kit comprising, in suitable container means, an expression vector comprising a humanized GFP gene.