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.

1 . A gene expression construct comprising:
 (a) an expression enhancer sequence derived from the RNA-2 genome segment of a Comoviridae bipartite RNA virus, in which a target initiation site in the RNA-2 genome segment has been mutated,
 wherein the wild-type RNA2 genome segment of the Comoviridae virus encodes two carboxy coterminal proteins through two different translation initiation sites located in the same triplet reading frame, and wherein the mutated initiation site is the first of these two initiation sites and thus corresponds to the initiation site at position 161 in the wild-type RNA-2 segment of cowpea mosaic virus (CPMV), 
 wherein the expression enhancer sequence comprises at least a 100 nucleotide sequence of the comoviral RNA-2 genome segment which includes the mutated initiation site; and 
   (b) a heterologous sequence for facilitating insertion of a gene encoding a protein of interest into the gene expression construct, wherein the heterologous sequence is located downstream of the mutated target initiation site in the enhancer sequence; and optionally   (c) a 3′ UTR.   
     
     
         2 . A gene expression system comprising an expression construct according to  claim 1 . 
     
     
         3 . A gene expression system comprising:
 (a) an expression enhancer sequence derived from the RNA-2 genome segment of a Comoviridae bipartite RNA virus, in which a target initiation site in the RNA-2 genome segment has been mutated,
 wherein the wild-type RNA2 genome segment of the Comoviridae virus encodes two carboxy coterminal proteins through two different translation initiation sites located in the same triplet reading frame, and wherein the mutated initiation site is the first of these two initiation sites and thus corresponds to the initiation site at position 161 in the wild-type RNA-2 segment of CPMV 
 wherein the expression enhancer sequence comprises at least a 100 nucleotide sequence of the comoviral RNA-2 genome segment which includes the mutated initiation site; and 
   (b) a heterologous gene encoding a protein of interest, wherein the gene encoding the protein of interest is located downstream of the enhancer sequence.   
     
     
         4 . A gene expression system according to  claim 3 , wherein the gene encoding the protein of interest is operably linked to promoter and terminator sequences. 
     
     
         5 . A gene expression system according to  claim 4 , wherein the gene encoding the protein of interest is located downstream of the enhancer sequence and upstream of the terminator sequence. 
     
     
         6 . A gene expression system according to  claim 3  comprising or further comprising a 3′ UTR which is optionally derived from the same bipartite RNA virus. 
     
     
         7 . A gene expression system according to  claim 3 , wherein the bipartite RNA virus is a comovirus. 
     
     
         8 . A gene expression system according to  claim 7 , wherein the comovirus is CPMV. 
     
     
         9 . A gene expression system according to  claim 7 , wherein the enhancer sequence comprises at least nucleotides 10 to 512, 20 to 512, 30 to 512, 40 to 512, 50 to 512, 100 to 512, 150 to 512, 1 to 514, 10 to 514, 20 to 514, 30 to 514, 40 to 514, 50 to 514, 100 to 514, 150 to 514, 1 to 511, 10 to 511, 20 to 511, 30 to 511, 40 to 511, 50 to 511, 100 to 511, 150 to 511, 1 to 509, 10 to 509, 20 to 509, 30 to 509, 40 to 509, 50 to 509, 100 to 509, 150 to 509, 1 to 507, 10 to 507, 20 to 507, 30 to 507, 40 to 507, 50 to 507, 100 to 507, or 150 to 507 of a comoviral RNA-2 genome segment sequence with said mutated target initiation site. 
     
     
         10 . A gene expression system according to  claim 8  wherein the enhancer sequence comprises nucleotides 10 to 512, 20 to 512, 30 to 512, 40 to 512, 50 to 512, 100 to 512, 150 to 512, 1 to 514, 10 to 514, 20 to 514, 30 to 514, 40 to 514, 50 to 514, 100 to 514, 150 to 514, 1 to 511, 10 to 511, 20 to 511, 30 to 511, 40 to 511, 50 to 511, 100 to 511, 150 to 511, 1 to 509, 10 to 509, 20 to 509, 30 to 509, 40 to 509, 50 to 509, 100 to 509, 150 to 509, 1 to 507, 10 to 507, 20 to 507, 30 to 507, 40 to 507, 50 to 507, 100 to 507, or 150 to 507 of the CPMV RNA-2 genome segment sequence shown in Table 1, wherein the target initiation site at position 161 in the wild-type CPMV RNA-2 genome segment has been mutated. 
     
     
         11 . A gene expression construct according to  claim 8  wherein the AUG at position 115 is also mutated. 
     
     
         12 . A gene expression construct according to  claim 3  further encoding a heterologous suppressor of gene silencing operably linked to promoter and terminator sequences. 
     
     
         13 . A gene expression construct according to  claim 3  which is a DNA binary vector. 
     
     
         14 . A process for increasing the expression or translational enhancing activity of a sequence derived from an RNA-2 genome segment of a Comoviridae bipartite virus, comprising mutating a target initiation site therein,
 wherein the wild-type RNA2 genome segment of the Comoviridae virus encodes two carboxy coterminal proteins through two different translation initiation sites located in the same triplet reading frame, and wherein the mutated initiation site is the first of these two initiation sites and thus corresponds to the initiation site at position 161 in the wild-type RNA-2 segment of CPMV.   
     
     
         15 . A process according to  claim 14  wherein said mutating is performed either by:
 (i) a point mutation in the initiation site, or 
 (ii) a deletion of up to 50 nucleotides in length including said initiation site,
 wherein said mutation enhances the expression of a heterologous ORF to which the sequence is attached. 
 
 
     
     
         16 . A process according to  claim 14 , wherein the bipartite RNA virus is a comovirus. 
     
     
         17 . A process according to  claim 15 , wherein the comovirus is cowpea mosaic virus. 
     
     
         18 . A method for expressing a protein of interest in a host organism using a gene expression system according to  claim 3 . 
     
     
         19 . A method according to  claim 18 , wherein the host organism is a eukaryotic host, which is optionally a plant or in insect. 
     
     
         20 . A method of enhancing the translation of a heterologous protein of interest from a gene or open reading frame (ORF) encoding the same which is operably linked to an RNA2-derived sequence,
 wherein the wild-type RNA2 genome segment of the Comoviridae virus encodes two carboxy coterminal proteins through two different translation initiation sites located in the same triplet reading frame,   the method comprising mutating the first of these two initiation sites in the RNA2-derived sequence,   wherein the mutated initiation site corresponds to the initiation site at position 161 in the wild-type RNA-2 segment of CPMV,   wherein the RNA1-derived sequence comprises at least a 100 nucleotide sequence of the comoviral RNA-2 genome segment which includes the mutated initiation site.   
     
     
         21 . A gene expression system comprising:
 (a) a first gene construct comprising a sequence derived from a truncated RNA-2 of a Comoviridae bipartite virus genome carrying at least one foreign gene encoding a heterologous protein of interest operably linked to promoter and terminator sequences, wherein the gene construct comprises a mutated target initiation site upstream of the foreign gene,   wherein the wild-type RNA2 genome segment of the Comoviridae virus encodes two carboxy coterminal proteins through two different translation initiation sites located in the same triplet reading frame, and   wherein the mutated initiation site is the first of these two initiation sites and thus corresponds to the initiation site at position 161 in the wild-type RNA-2 segment of CPMV; and optionally   (b) a second gene construct optionally incorporated within said first gene construct comprising a heterologous suppressor of gene silencing operably linked to promoter and terminator sequences.   
     
     
         22 . A method of expressing a heterologous protein in a plant comprising the steps of:
 (a) introducing a gene expression construct comprising a first gene construct comprising a sequence derived from a truncated RNA-2 segment of a Comoviridae bipartite virus genome carrying at least one foreign gene encoding a heterologous protein of interest operably linked to promoter and terminator sequences, wherein the gene construct comprises a mutated target initiation site upstream of the foreign gene into a plant cell,   wherein the wild-type RNA2 genome segment of the Comoviridae virus encodes two carboxy coterminal proteins through two different translation initiation sites located in the same triplet reading frame,   wherein the mutated initiation site is the first of these two initiation sites and thus corresponds to the initiation site at position 161 in the wild-type RNA-2 segment of CPMV; and   (b) introducing a second gene construct optionally incorporated within said first gene construct, comprising a heterologous suppressor of gene silencing operably linked to promoter and terminator sequences into the plant cell.   
     
     
         23 . A method according to  claim 18 , comprising the step of introducing the gene expression system into the host organism. 
     
     
         24 . A host organism obtained or obtainable by a method according to  claim 23 , wherein the gene encoding the protein of interest is expressed at an enhanced level. 
     
     
         25 . A host organism transiently transfected with a gene expression system according to  claim 3 . 
     
     
         26 . A host organism according to  claim 24 , wherein the host organism is a plant or plant cell. 
     
     
         27 . A transgenic host organism stably transformed with a gene expression system according to  claim 3 . 
     
     
         28 . A method for generating a heterologous protein of interest, comprising using a host organism according to  claim 24  and optionally harvesting a tissue in which the protein of interest has been expressed and isolating the protein of interest from the tissue. 
     
     
         29 . A gene expression system according to  claim 3  wherein the gene encoding the protein of interest is a gene which modifies or imparts a characteristic selected from the list consisting of: an insect resistance; disease resistance; herbicide resistance; sterility; a grain characteristic selected which is oil, starch or carbohydrate content; and response to environmental- or other stress 
     
     
         30 . A gene expression system according to  claim 3  wherein the gene encoding the protein of interest is a gene responsible for the synthesis of a product selected from the list consisting of: proteins, peptides, fatty acids, lipids, waxes, oils, starches, sugars, carbohydrates, flavors, odors, toxins, carotenoids, hormones, polymers, flavonoids, storage proteins, phenolic acids, alkaloids, lignins, tannins, celluloses, glycoproteins, and glycolipids. 
     
     
         31 . A gene expression system according to  claim 3  wherein the protein of interest is a mammalian protein. 
     
     
         32 . A gene expression system according to  claim 3  wherein the expression enhancer sequence comprises at least a 200 nucleotide sequence of the comoviral RNA-2 genome segment which includes the mutated initiation site.