Protein expression systems

Abstract

The invention is based on an expression enhancer sequence derived from the RNA-2 genome segment of a bipartite RNA virus, in which a target initiation site in the RNA-2 genome segment has been mutated. Deletion of appropriate start codons upstream of the main RNA2 translation initiation can greatly increase in foreign protein accumulation without the need for viral replication. Also provided are methods, vectors and systems, including the ‘hyper-translatable’ Cowpea Mosaic Virus (“CPMV-HT”) based protein expression system.

What is claimed: 
     
         1 . A gene expression construct comprising:
 at least one fragment of the RNA-2 genome segment of Cowpea Mosaic Virus (CPMV) set forth in SEQ ID NO: 1,
 wherein the fragment of the RNA-2 genome segment of CPMV consists essentially of at least 100 contiguous nucleotides of SEQ ID NO:1, and does not contain the target initiation site at positions 161-163 of SEQ ID NO: 1 
   a coding sequence for a protein heterologous to Cowpea Mosaic Virus,
 wherein the coding sequence is operably linked to a functional translation initiation site, and 
   wherein the heterologous protein is expressed in a plant.   
     
     
         2 . The gene expression construct of  claim 1  consisting essentially of a sequence starting with nucleotide 1 of SEQ ID NO:1 and up to target initiation site at positions 161-163. 
     
     
         3 . The gene expression construct of  claim 1  consisting essentially of a mutated initiation site at positions 115-117, whereby the initiation site does not function as a translation initiation site. 
     
     
         4 . The gene expression construct of  claim 3 , wherein the codon of positions 115-117 of SEQ ID NO:1 is GUG. 
     
     
         5 . The gene expression construct of  claim 2  consisting essentially of a mutated initiation site at positions 115-117, whereby the initiation site does not function as a translation initiation site. 
     
     
         6 . The gene expression construct of  claim 5 , wherein the codon of positions 115-117 of SEQ ID NO:1 is GUG. 
     
     
         7 . A gene expression construct comprising:
 at least one fragment of the RNA-2 genome segment of Cowpea Mosaic Virus (CPMV) set forth in SEQ ID NO: 1,
 wherein the fragment of the RNA-2 genome segment of CPMV consists essentially of a sequence starting with nucleotide 1 of SEQ ID NO:1 and up to target initiation site at positions 161-163, and the target initiation site at positions 161-163 of SEQ ID NO: 1 is mutated and does not function as a translation initiation site, 
   a coding sequence for a protein heterologous to Cowpea Mosaic Virus,
 wherein the coding sequence is operably linked to a functional translation initiation site, and 
   wherein the heterologous protein is expressed in a plant.   
     
     
         8 . The gene expression construct of  claim 7 , wherein the target initiation site at positions 161-163 of SEQ ID NO: 1 is deleted. 
     
     
         9 . The gene expression construct of  claim 7  consisting essentially of a mutated initiation site at positions 115-117, whereby the initiation site does not function as a translation initiation site. 
     
     
         10 . The gene expression construct of  claim 9 , wherein the codon of positions 115-117 of SEQ ID NO:1 is GUG. 
     
     
         11 . The gene expression construct of  claim 6  consisting essentially of a mutated initiation site at positions 115-117, whereby the initiation site does not function as a translation initiation site. 
     
     
         12 . The gene expression construct of  claim 11 , wherein the codon of positions 115-117 of SEQ ID NO:1 is GUG. 
     
     
         13 . A method for expressing a protein heterologous to CPMV in a plant comprising:
 introducing the gene expression construct of  claim 1  into a plant cell of the plant,   whereby the heterologous protein is expressed in a plant.   
     
     
         14 . A method for expressing a protein heterologous to CPMV in a plant comprising:
 introducing the gene expression construct of  claim 2  into a plant cell of the plant,   whereby the heterologous protein is expressed in a plant.   
     
     
         15 . A method for expressing a protein heterologous to CPMV in a plant comprising:
 introducing the gene expression construct of  claim 3  into a plant cell of the plant,   whereby the heterologous protein is expressed in a plant.   
     
     
         16 . A method for expressing a protein heterologous to CPMV in a plant comprising:
 introducing the gene expression construct of  claim 5  into a plant cell of the plant,   whereby the heterologous protein is expressed in a plant.   
     
     
         17 . A method for expressing a protein heterologous to CPMV in a plant comprising:
 introducing the gene expression construct of  claim 7  into a plant cell of the plant,   whereby the heterologous protein is expressed in a plant.   
     
     
         18 . A method for expressing a protein heterologous to CPMV in a plant comprising:
 introducing the gene expression construct of  claim 8  into a plant cell of the plant,   whereby the heterologous protein is expressed in a plant.   
     
     
         19 . A method for expressing a protein heterologous to CPMV in a plant comprising:
 introducing the gene expression construct of  claim 9  into a plant cell of the plant,   whereby the heterologous protein is expressed in a plant.   
     
     
         20 . A method for expressing a protein heterologous to CPMV in a plant comprising:
 introducing the gene expression construct of  claim 11  into a plant cell of the plant,   whereby the heterologous protein is expressed in a plant.   
     
     
         21 . The method of  claim 13 , further comprising introducing into the plant cell a heterologous suppressor of gene silencing. 
     
     
         22 . The method of  claim 14 , further comprising introducing into the plant cell a heterologous suppressor of gene silencing. 
     
     
         23 . The method of  claim 15 , further comprising introducing into the plant cell a heterologous suppressor of gene silencing. 
     
     
         24 . The method of  claim 16 , further comprising introducing into the plant cell a heterologous suppressor of gene silencing. 
     
     
         25 . The method of  claim 17 , further comprising introducing into the plant cell a heterologous suppressor of gene silencing. 
     
     
         26 . The method of  claim 18 , further comprising introducing into the plant cell a heterologous suppressor of gene silencing. 
     
     
         27 . The method of  claim 19 , further comprising introducing into the plant cell a heterologous suppressor of gene silencing. 
     
     
         28 . The method of  claim 20 , further comprising introducing into the plant cell a heterologous suppressor of gene silencing. 
     
     
         29 . The method of  claim 13 , further comprising harvesting the heterologous protein. 
     
     
         30 . The method of  claim 14 , further comprising harvesting the heterologous protein. 
     
     
         31 . The method of  claim 15 , further comprising harvesting the heterologous protein. 
     
     
         32 . The method of  claim 16 , further comprising harvesting the heterologous protein. 
     
     
         33 . The method of  claim 17 , further comprising harvesting the heterologous protein. 
     
     
         34 . The method of  claim 18 , further comprising harvesting the heterologous protein. 
     
     
         35 . The method of  claim 19 , further comprising harvesting the heterologous protein. 
     
     
         36 . The method of  claim 20 , further comprising harvesting the heterologous protein. 
     
     
         37 . The method of  claim 21 , further comprising harvesting the heterologous protein. 
     
     
         38 . The method of  claim 22 , further comprising harvesting the heterologous protein. 
     
     
         39 . The method of  claim 23 , further comprising harvesting the heterologous protein. 
     
     
         40 . The method of  claim 24 , further comprising harvesting the heterologous protein. 
     
     
         41 . The method of  claim 25 , further comprising harvesting the heterologous protein. 
     
     
         42 . The method of  claim 26 , further comprising harvesting the heterologous protein. 
     
     
         43 . The method of  claim 27 , further comprising harvesting the heterologous protein. 
     
     
         44 . The method of  claim 28 , further comprising harvesting the heterologous protein. 
     
     
         45 . The method of  claim 29  further comprising preparing an extract of the heterologous protein. 
     
     
         46 . The method of  claim 30  further comprising preparing an extract of the heterologous protein. 
     
     
         47 . The method of  claim 31  further comprising preparing an extract of the heterologous protein. 
     
     
         48 . The method of  claim 32  further comprising preparing an extract of the heterologous protein. 
     
     
         49 . The method of  claim 33  further comprising preparing an extract of the heterologous protein. 
     
     
         50 . The method of  claim 34  further comprising preparing an extract of the heterologous protein. 
     
     
         51 . The method of  claim 35  further comprising preparing an extract of the heterologous protein. 
     
     
         52 . The method of  claim 36  further comprising preparing an extract of the heterologous protein. 
     
     
         53 . The method of  claim 37  further comprising preparing an extract of the heterologous protein. 
     
     
         54 . The method of  claim 38  further comprising preparing an extract of the heterologous protein. 
     
     
         55 . The method of  claim 39  further comprising preparing an extract of the heterologous protein. 
     
     
         56 . The method of  claim 40  further comprising preparing an extract of the heterologous protein. 
     
     
         57 . The method of  claim 41  further comprising preparing an extract of the heterologous protein. 
     
     
         58 . The method of  claim 42  further comprising preparing an extract of the heterologous protein. 
     
     
         59 . The method of  claim 43  further comprising preparing an extract of the heterologous protein. 
     
     
         60 . The method of  claim 44  further comprising preparing an extract of the heterologous protein.