RNA binding protein and binding site useful for expression of recombinant molecules

Abstract

The present invention relates to a gene expression system in eukaryotic and prokaryotic cells, preferably plant cells and intact plants. In particular, the invention relates to an expression system having a RB47 binding site upstream of a translation initiation site for regulation of translation mediated by binding of RB47 protein, a member of the poly(A) binding protein family. Regulation is further effected by RB60, a protein disulfide isomerase. The expression system is capable of functioning in the nuclear/cytoplasm of cells and in the chloroplast of plants. Translation regulation of a desired molecule is enhanced approximately 100 fold over that obtained without RB47 binding site activation.

1. An expression cassette for expression of a desired molecule, which cassette comprises:
 a) an RB47 binding site nucleotide sequence upstream of a restriction endonuclease site for insertion of a desired coding sequence to be expressed; and  
 b) a nucleotide sequence encoding a polypeptide which binds RB47 binding site.  
 
     
     
       2. The expression cassette of  claim 1  further comprising a promoter sequence operably linked to and positioned upstream of the RB47 binding site nucleotide sequence. 
     
     
       3. The expression cassette of  claim 2  wherein the promoter sequence is derived from a psbA gene. 
     
     
       4. The expression cassette of  claim 3  wherein the coding sequence is heterologous to the psbA gene. 
     
     
       5. The expression cassette of  claim 1  wherein the cassette comprises a plasmid or virus. 
     
     
       6. The expression cassette of  claim 1  further comprising and operably linked thereto a nucleotide sequence encoding RB60. 
     
     
       7. The expression cassette of  claim 1  wherein the RB47 binding polypeptide is selected from the group consisting of RB47, RB47 precursor and a histidine-modified RB47. 
     
     
       8. An expression cassette for expression of a desired molecule, which cassette comprises:
 a) an RB47 binding site nucleotide sequence upstream of a restriction endonuclease site for insertion of a desired coding sequence to be expressed,  
 
       and
 b) a nucleotide sequence encoding a polypeptide which regulates the binding of RB47 to the RB47 binding site.  
 
     
     
       9. The expression cassette of  claim 8  wherein the regulatory polypeptide is RB60. 
     
     
       10. A method of screening for agonists or antagonists of RB47 binding to RB47 binding site, the method comprising the steps:
 a) providing a cell expression system containing 
 1) a promoter sequence,  
 2) a RB47 binding site sequence;  
 3) a coding sequence for an indicator polypeptide; and  
 4) a polypeptide which binds to the RB47 binding site sequence;  
 
 b) introducing an antagonist or agonist into the cell; and  
 c) detecting the amount of indicator polypeptide expressed in the cell.  
 
     
     
       11. A method of screening for agonists or antagonists of RB60 in regulating RB47 binding to RB47 binding site, the method comprising the steps:
 a) providing an expression system in a cell containing: 
 1) a promoter sequence;  
 2) a RB47 binding site sequence;  
 3) a coding sequence for an indicator polypeptide;  
 4) a polypeptide which binds to the RB47 binding site sequence, and  
 5) a RB60 polypeptide;  
 
 b) introducing an agonist or antagonist into the cell; and  
 c) detecting the amount of indicator polypeptide expressed in the cell.  
 
     
     
       12. An isolated nucleotide sequence encoding RB47. 
     
     
       13. An isolated nucleotide sequence encoding a histidine-modified RB47. 
     
     
       14. An isolated nucleotide sequence encoding RB47 precursor. 
     
     
       15. The nucleotide sequence of  claim 12  from nucleotide position 197 to 1402 in  FIGS. 1A-1B  and SEQ ID NO 5. 
     
     
       16. The nucleotide sequence of  claim 13  from nucleotide position 1 to 1269 in  FIGS. 5A-5B  and SEQ ID NO 14. 
     
     
       17. The nucleotide sequence of  claim 14  shown in from nucleotide position 197 to 2065 in  FIGS. 1A-1C  and SEQ ID NO 5. 
     
     
       18. An expression cassette comprising the nucleotide sequence of  claim 12 ,  13  or  14 . 
     
     
       19. An isolated nucleotide sequence encoding RB60. 
     
     
       20. The nucleotide sequence of  claim 18  from nucleotide position 16 to 1614 in  FIGS. 2A-2B  and SEQ ID NO 10. 
     
     
       21. An expression cassette comprising the nucleotide sequence of  claim 19 . 
     
     
       22. An expression system comprising a cell transformed with the expression cassette of  claim 1 . 
     
     
       23. The expression system of  claim 22  wherein the cell is a plant cell. 
     
     
       24. The expression system of  claim 23  wherein the plant cell endogenously expresses RB47. 
     
     
       25. The expression system of  claim 23  wherein the plant cell endogenously expresses RB60. 
     
     
       26. The expression system of  claim 23  wherein the plant cell endogenously expresses RB47 and RB60. 
     
     
       27. The expression system of  claim 22  wherein the cell is a eukaryotic cell. 
     
     
       28. The expression system of  claim 22  wherein the cell is a prokaryotic cell. 
     
     
       29. The expression system of  claim 22  further comprising the an expression cassette of  claim 21  comprising an isolated nucleotide sequence encoding RB 60 . 
     
     
       30. An expression system comprising a cell transformed with the expression cassette of  claim 8 . 
     
     
       31. The expression system of  claim 29  further comprising the an expression cassette of  claim 18  comprising an isolated nucleotide sequence encoding RB 47 , a histidine- modified RB 47 , or RB 47  precursor . 
     
     
       32. A cell stably transformed with the expression cassette of  claim 18 . 
     
     
       33. A cell stably transformed with the expression cassette of  claim 21 . 
     
     
       34. A cell stably transformed with the an expression cassette of claims  18  and  21  comprising an isolated nucleotide sequence encoding RB 47 , histidine- modified RB 47 , RB 47  precursor, or RB 60   . 
     
     
       35. The expression cassette of  claim 1  further comprising an inserted desired coding sequence. 
     
     
       36. An expression system comprising a cell transformed with the expression cassette of  claim 35 , wherein the coding sequence is expressed forming the desired molecule upon activation of the RB47 binding site with RB47. 
     
     
       37. The expression system of  claim 36  wherein the cell is a plant cell endogenously expressing RB47. 
     
     
       38. The expression system of  claim 36  wherein the cell is stably transformed with the an expression cassette of  claim 21  comprising an isolated nucleotide sequence encoding RB 60 . 
     
     
       39. An expression system comprising a cell transformed with an expression cassette comprising a promoter sequence, a RB47 binding site sequence, a desired coding sequence for a molecule, and a nucleotide sequence for encoding a polypeptide which binds RB47 binding site, wherein all sequences are operably linked. 
     
     
       40. A method of preparing a desired recombinant molecule wherein the method comprises cultivating the expression system of  claim 36 . 
     
     
       41. A method of preparing a desired recombinant molecule wherein the method comprises cultivating the expression system of  claim 39 . 
     
     
       42. A method for expressing a desired coding sequence comprising:
 a) forming an expression cassette by operably linking: 
 1) a promoter sequence;  
 2) a RB47 binding site sequence;  
 3) a desired coding sequence; and  
 4) a nucleotide sequence encoding a polypeptide which binds RB47 binding site, and  
 
 b) introducing the expression cassette into a cell.  
 
     
     
       43. The method of  claim 42  wherein the cell is a plant cell endogenously expressing RB47. 
     
     
       44. The method of  claim 42  wherein the cell is a plant cell endogenously expressing RB60. 
     
     
       45. The method of  claim 42  further comprising inducing expression with a promoter inducer molecule. 
     
     
       46. The method of  claim 45  wherein the promoter inducer molecule is IPTG. 
     
     
       47. The method of  claim 42  wherein the cell is transformed with the an expression cassette of  claim 21  comprising an isolated nucleotide sequence encoding RB 60 . 
     
     
       48. A method for expressing a desired coding sequence comprising:
 a) forming an expression cassette by operably linking: 
 1) a promoter sequence;  
 2) a RB47 binding site sequence; and  
 3) a desired coding sequence;  
 
 
       and
 b) introducing the expression cassette into a plant cell endogenously expressing RB47.  
 
     
     
       49. The method of  claim 48  wherein the expression cassette further comprises a nucleotide sequence encoding RB60. 
     
     
       50. A method for the regulated production of a recombinant molecule from a desired coding sequence in a cell, wherein the cell contains the expression cassette of  claim 34 , wherein expression of the coding sequence is activated by RB47 binding to the RB47 binding site thereby producing the recombinant molecule. 
     
     
       51. A method of forming an expression cassette by operably linking:
 a) a RB47 binding site sequence;  
 b) a cloning site for insertion of a desired coding sequence downstream of the RB47 binding site sequence; and  
 c) a nucleotide sequence encoding a polypeptide which binds the RB47 binding site.  
 
     
     
       52. The method of  claim 51  further comprising a promoter sequence operably linked upstream to the RB47 binding site sequence. 
     
     
       53. The method of  claim 51  further comprising a desired coding sequence inserted into the insertion site. 
     
     
       54. An article of manufacture comprising a packaging material and contained therein in a separate container the expression cassette of  claim 1 , wherein the expression cassette is useful for expression of a desired coding sequence, and wherein the packaging material comprises a label which indicates that the expression cassette can be used for expressing a desired coding sequence when the RB47 binding is activated by RB47. 
     
     
       55. The article of manufacture of  claim 54  further comprising in a separate container the an expression cassette of  claim 18  comprising an isolated nucleotide sequence encoding RB 47 , a histidine- modified RB 47 , or RB 47  precursor . 
     
     
       56. The article of manufacture of  claim 54  further comprising in a separate container the an expression cassette of  claim 21  comprising an isolated nucleotide sequence encoding RB 60 . 
     
     
       57. An article of manufacture comprising a packaging material and contained therein in a separate container the expression system of  claim 22 , wherein the expression system is useful for expression of a desired coding sequence, and wherein the packaging material comprises a label which indicates that the expression system can be used for expressing a desired coding sequence when the RB47 binding site is activated by RB47. 
     
     
       58. An article of manufacture comprising a packaging material and contained therein in a separate container the stably transformed cell of  claim 32 , wherein the cell is useful as an expression system, and wherein the packaging material comprises a label which indicates that the expression system can be used for expressing a desired coding sequence when the RB47 binding site is activated by RB47. 
     
     
       59. An article of manufacture comprising a packaging material and contained therein in a separate container the stably transformed cell of  claim 33 , wherein the cell is useful as an expression system, and wherein the packaging material comprises a label which indicates that the expression system can be used for expressing a desired coding sequence when the RB47 binding site is activated by RB47 and regulated by RB60. 
     
     
       60. An article of manufacture comprising a packaging material and contained therein in a separate container the stably transformed cell of  claim 34 , wherein the cell is useful as an expression system, and wherein the packaging material comprises a label which indicates that the expression system can be used for expressing a desired coding sequence when the RB47 binding site is activated by RB47 and regulated by RB60. 
     
     
       61. An article of manufacture comprising a packaging material and contained therein in a separate container the expression cassette of  claim 2 , wherein the expression cassette is useful for expression of a RNA transcript, and wherein the packaging material comprises a label which indicates that the expression cassette can be used for producing in vitro a RNA transcript when the RB47 binding site is activated by RB47. 
     
     
       62. The article of manufacture of  claim 61  wherein the promoter sequence is selected from the group consisting of T3 and T7 promoters. 
     
     
       63. The article of manufacture of  claim 61  further comprising in separate containers a polymerase, a buffer and each of four ribonucleotides, reagents for in vitro RNA transcription. 
     
     
       64. An expression cassette for the expression of a desired eukaryotic molecule within a plastid comprising a suitable promoter operably linked to a eukaryotic transgene of interest, wherein said eukaryotic molecule comprises an antibody. 
     
     
       65. The expression cassette of  claim 64 , wherein the promoter is a homologous promoter. 
     
     
       66. The expression cassette of  claim 64 , wherein the promoter is a psbA promoter. 
     
     
       67. The expression cassette of  claim 64 , further comprising a  5 ′ UTR. 
     
     
       68. The expression cassette of  claim 67 , wherein the  5 ′UTR further comprises a RB 47  binding site sequence. 
     
     
       69. The expression cassette of  claim 67 , further comprising a  3 ′ UTR. 
     
     
       70. The expression cassette of  claim 64 , wherein the plastid comprises a chloroplast. 
     
     
       71. The expression cassette of  claim 64 , wherein the antibody is a single chain antibody. 
     
     
       72. The expression cassette of  claim 64 , wherein the antibody is a dimeric antibody. 
     
     
       73. The expression cassette of  claim 64 , wherein the expression cassette further encodes a luciferase enzyme. 
     
     
       74. The expression cassette of  claim 64 , wherein the promoter is constitutive. 
     
     
       75. The expression cassette of  claim 64 , wherein the promoter is inducible. 
     
     
       76. The expression cassette of  claim 64 , wherein the promoter is a eukaryotic promoter. 
     
     
       77. The expression cassette of  claim 64 , wherein the promoter is a prokaryotic promoter. 
     
     
       78. The expression cassette of  claim 64 , further comprising an origin of replication. 
     
     
       79. The expression cassette of  claim 64 , further comprising a selectable marker. 
     
     
       80. A cell comprising the expression cassette of  claim 64 . 
     
     
       81. The cell of  claim 80 , wherein the cell is a plant cell. 
     
     
       82. The cell of  claim 81 , wherein the plant cell comprises a plastid. 
     
     
       83. The cell of  claim 82 , wherein the plastid comprises a chloroplast. 
     
     
       84. The cell of  claim 81 , wherein the plant cell comprises a mitochondria. 
     
     
       85. The cell of  claim 80 , wherein the cell is an algae cell. 
     
     
       86. The cell of  claim 85 , wherein the cell is a Chlamydomonas reinhardtii cell. 
     
     
       87. A method for producing a eukaryotic protein of interest comprising transforming a plastid with the expression cassette of  claim 64 , allowing the cell to grow, and harvesting the protein. 
     
     
       88. The method of  claim 87 , wherein the plastid is comprised within a plant cell. 
     
     
       89. The method of  claim 87 , wherein the plastid is comprised within an algae cell. 
     
     
       90. The method of  claim 87 , wherein the transformation occurs in vitro. 
     
     
       91. The method of  claim 87 , wherein the transformation occurs in vivo. 
     
     
       92. The method of  claim 87 , wherein the transformation occurs ex vivo. 
     
     
       93. The expression cassette of  claim 64 , wherein the promoter is a heterologous promoter. 
     
     
       94. The expression cassette of  claim 64 , wherein the promoter is a bacterial promoter, bacteriophage promoter, T 3  promoter or a T 7  promoter. 
     
     
       95. The expression cassette of  claim 64 , wherein the promoter is a constitutive promoter or an inducible promoter. 
     
     
       96. A DNA construct for expression of a transgene within a plastid comprising a promoter functional in a plastid operably linked to a gene of interest, wherein said transgene comprises a gene encoding an antibody. 
     
     
       97. The expression cassette of  claim 65 , further comprising a  5 ′ UTR. 
     
     
       98. The expression cassette of  claim 65 , wherein the plastid comprises a chloroplast. 
     
     
       99. The expression cassette of  claim 65 , further comprising an origin of replication. 
     
     
       100. The expression cassette of  claim 65 , further comprising a selectable marker.