Methods and compositions for high yield production of eukaryotic proteins

Abstract

The present invention provides an isolated nucleic acid comprising a first nucleotide sequence encoding an amino acid sequence comprising at least three positively charged amino acid residues, positioned upstream and in frame with a second nucleotide sequence encoding a protein. In addition, the present invention provides an isolated nucleic acid comprising a first nucleotide sequence encoding a DNA binding protein, positioned upstream and in frame with a second nucleotide sequence encoding a protein. An isolated nucleic acid is also provided, which comprises a first nucleotide sequence encoding a bacteriophage lambda repressor protein, positioned upstream and in frame with a second nucleotide sequence encoding a protein. The present invention further provides a method of producing a eukaryotic protein in a bacterial cell comprising: a) introducing the expression vector of this invention, wherein the second nucleotide sequence encodes a eukaryotic protein, into the bacterial cell; and b) culturing the bacterial cell under conditions whereby the second nucleotide sequence of the expression vector is expressed to produce the eukaryotic protein. A method of producing a eukaryotic protein in a bacterial cell in high yield is also provided, comprising: a) introducing the expression vector of this invention, wherein the second nucleotide sequence encodes a eukaryotic protein, into the bacterial cell; and b) culturing the bacterial cell under conditions whereby the second nucleotide sequence of the expression vector is expressed to produce the eukaryotic protein in high yield.

What is claimed is:  
     
         1 . An isolated nucleic acid comprising a first nucleotide sequence encoding an amino acid sequence comprising at least three positively charged amino acid residues, positioned upstream and in frame with a second nucleotide sequence encoding a protein.  
     
     
         2 . An isolated nucleic acid comprising a first nucleotide sequence encoding a DNA binding protein, positioned upstream and in frame with a second nucleotide sequence encoding a protein.  
     
     
         3 . The nucleic acid of  claim 2 , wherein the DNA binding protein is selected from the group consisting of eukaryotic DNA binding proteins, prokaryotic DNA binding proteins and bacteriophage-derived DNA binding proteins.  
     
     
         4 . An isolated nucleic acid comprising a first nucleotide sequence encoding a bacteriophage lambda repressor protein, positioned upstream and in frame with a second nucleotide sequence encoding a protein.  
     
     
         5 . The nucleic acid of  claim 4 , wherein the first nucleotide sequence encodes the N-terminal domain of the bacteriophage lambda repressor protein.  
     
     
         6 . The nucleic acid of  claim 4 , wherein the first nucleotide sequence encodes amino acids 1-76 of the bacteriophage lambda repressor protein.  
     
     
         7 . The nucleic acid of  claim 4 , wherein the first nucleotide sequence encodes at least 15 contiguous amino acids of the N-terminus of the bacteriophage lambda repressor protein.  
     
     
         8 . The nucleic acid of  claim 1 , wherein the second nucleotide sequence encodes a protein selected from the group consisting of integral membrane proteins, G-protein coupled receptor proteins and ion channel proteins.  
     
     
         9 . The nucleic acid of  claim 2 , wherein the second nucleotide sequence encodes a protein selected from the group consisting of integral membrane proteins, G-protein coupled receptor proteins and ion channel proteins.  
     
     
         10 . The nucleic acid of  claim 3 , wherein the second nucleotide sequence encodes a protein selected from the group consisting of integral membrane proteins, G-protein coupled receptor proteins and ion channel proteins.  
     
     
         11 . The nucleic acid of  claim 4 , wherein the second nucleotide sequence encodes a protein selected from the group consisting of integral membrane proteins, G-protein coupled receptor proteins and ion channel proteins.  
     
     
         12 . The nucleic acid of  claim 5 , wherein the second nucleotide sequence encodes a protein selected from the group consisting of integral membrane proteins, G-protein coupled receptor proteins and ion channel proteins.  
     
     
         13 . The nucleic acid of  claim 6 , wherein the second nucleotide sequence encodes a protein selected from the group consisting of integral membrane proteins, G-protein coupled receptor proteins and ion channel proteins.  
     
     
         14 . The nucleic acid of  claim 7 , wherein the second nucleotide sequence encodes a protein selected from the group consisting of integral membrane proteins, G-protein coupled receptor proteins and ion channel proteins.  
     
     
         15 . The nucleic acid of  claim 1 , wherein the second nucleotide sequence encodes a protein selected from the group consisting of rabbit prostaglandin E 2 EP 3  receptor protein, human prostaglandin E 2 EP 2  receptor protein, human chemokine receptor CCR-5 protein, human β 2  adrenergic receptor protein, rat renal outer medullary K +  channel protein and human small G-protein rho.  
     
     
         16 . The nucleic acid of  claim 2 , wherein the second nucleotide sequence encodes a protein selected from the group consisting of rabbit prostaglandin E 2 EP 3  receptor protein, human prostaglandin E 2 EP 2  receptor protein, human chemokine receptor CCR-5 protein, human β 2  adrenergic receptor protein, rat renal outer medullary K +  channel protein and human small G-protein rho.  
     
     
         17 . The nucleic acid of  claim 3 , wherein the second nucleotide sequence encodes a protein selected from the group consisting of rabbit prostaglandin E 2 EP 3  receptor protein, human prostaglandin E 2 EP 2  receptor protein, human chemokine receptor CCR-5 protein, human β 2  adrenergic receptor protein, rat renal outer medullary K +  channel protein and human small G-protein rho.  
     
     
         18 . The nucleic acid of  claim 4 , wherein the second nucleotide sequence encodes a protein selected from the group consisting of rabbit prostaglandin E 2 EP 3  receptor protein, human prostaglandin E 2 EP 2  receptor protein, human chemokine receptor CCR-5 protein, human β 2  adrenergic receptor protein, rat renal outer medullary K +  channel protein and human small G-protein rho.  
     
     
         19 . The nucleic acid of  claim 5 , wherein the second nucleotide sequence encodes a protein selected from the group consisting of rabbit prostaglandin E 2 EP 3  receptor protein, human prostaglandin E 2 EP 2  receptor protein, human chemokine receptor CCR-5 protein, human β 2  adrenergic receptor protein, rat renal outer medullary K +  channel protein and human small G-protein rho.  
     
     
         20 . The nucleic acid of  claim 6 , wherein the second nucleotide sequence encodes a protein selected from the group consisting of rabbit prostaglandin E 2 EP 3  receptor protein, human prostaglandin E 2 EP 2  receptor protein, human chemokine receptor CCR-5 protein, human β 2  adrenergic receptor protein, rat renal outer medullary K +  channel protein and human small G-protein rho.  
     
     
         21 . The nucleic acid of  claim 7 , wherein the second nucleotide sequence encodes a protein selected from the group consisting of rabbit prostaglandin E 2 EP 3  receptor protein, human prostaglandin E 2 EP 2  receptor protein, human chemokine receptor CCR-5 protein, human β 2  adrenergic receptor protein, rat renal outer medullary K +  channel protein and human small G-protein rho.  
     
     
         22 . The nucleic acid of  claim 8 , wherein the second nucleotide sequence encodes a protein selected from the group consisting of rabbit prostaglandin E 2 EP 3  receptor protein, human prostaglandin E 2 EP 2  receptor protein, human chemokine receptor CCR-5 protein, human β 2  adrenergic receptor protein, rat renal outer medullary K +  channel protein and human small G-protein rho.  
     
     
         23 . An isolated nucleic acid having the nucleotide sequence selected from the group consisting of SEQ ID NO:1 (rabbit prostaglandin E 2 EP 3  receptor protein), SEQ ID NO:2 (human prostaglandin E 2 EP 2  receptor protein), SEQ ID NO:3 (human chemokine receptor CCR-5 protein), SEQ ID NO:4 (human β 2  adrenergic receptor protein), SEQ ID NO:5 (rat renal outer medullary K +  channel protein) and SEQ ID NO:6 (human small G-protein rho).  
     
     
         24 . A method of producing a eukaryotic protein in a bacterial cell comprising: 
 a) introducing the nucleic acid of  claim 1 , wherein the second nucleotide sequence encodes a eukaryotic protein, into the bacterial cell; and    b) culturing the bacterial cell under conditions whereby the second nucleotide sequence of the nucleic acid is expressed to produce the eukaryotic protein.    
     
     
         25 . A method of producing a eukaryotic protein in a bacterial cell in high yield comprising: 
 a) introducing the nucleic acid of  claim 1 , wherein the second nucleotide sequence encodes a eukaryotic protein, into the bacterial cell; and    b) culturing the bacterial cell under conditions whereby the second nucleotide sequence of the nucleic acid is expressed to produce the eukaryotic protein in high yield.    
     
     
         26 . A method of producing a eukaryotic integral membrane protein in a bacterial cell comprising: 
 a) introducing the nucleic acid of  claim 1 , wherein the second nucleotide sequence encodes a eukaryotic integral membrane protein, into the bacterial cell; and    b) culturing the bacterial cell under conditions whereby the second nucleotide sequence of the nucleic acid is expressed to produce the eukaryotic integral membrane protein.    
     
     
         27 . A method of producing a eukaryotic G-protein coupled receptor protein in a bacterial cell comprising: 
 a) introducing the nucleic acid of  claim 1 , wherein the second nucleotide sequence encodes a eukaryotic G-protein coupled receptor protein, into the bacterial cell; and    b) culturing the bacterial cell under conditions whereby the second nucleotide sequence of the nucleic acid is expressed to produce the eukaryotic G-protein coupled receptor protein.    
     
     
         28 . A method of producing a eukaryotic ion channel protein in a bacterial cell comprising: 
 a) introducing the nucleic acid of  claim 1 , wherein the second nucleotide sequence encodes a eukaryotic ion channel protein, into the bacterial cell; and    b) culturing the bacterial cell under conditions whereby the second nucleotide sequence of the nucleic acid is expressed to produce the eukaryotic ion channel protein.    
     
     
         29 . A method of producing a rabbit prostaglandin E 2  EP 3  receptor protein in a bacterial cell comprising: 
 a) introducing the nucleic acid of  claim 1 , wherein the second nucleotide sequence encodes the rabbit prostaglandin E 2  EP 3  receptor protein, into the bacterial cell; and    b) culturing the cell under conditions whereby the second nucleotide sequence of the expression vector is expressed to produce the rabbit prostaglandin E 2  EP 3  receptor protein.    
     
     
         30 . A method of producing a human prostaglandin E 2  EP 2  receptor protein in a bacterial cell comprising: 
 a) introducing the nucleic acid of  claim 1 , wherein the second nucleotide sequence encodes the human prostaglandin E 2  EP 2  receptor protein, into the bacterial cell; and    b) culturing the bacterial cell under conditions whereby the second nucleotide sequence of the nucleic acid is expressed to produce the human prostaglandin E 2  EP 2  receptor protein.    
     
     
         31 . A method of producing a human chemokine receptor CCR-5 protein in a bacterial cell comprising: 
 a) introducing the nucleic acid of  claim 1 , wherein the second nucleotide sequence encodes the human chemokine receptor CCR-5 protein, into the bacterial cell; and    b) culturing the bacterial cell under conditions whereby the second nucleotide sequence of the nucleic acid is expressed to produce the human chemokine receptor CCR-5 protein.    
     
     
         32 . A method of producing a human β 2  adrenergic receptor protein in a bacterial cell comprising: 
 a) introducing the nucleic acid of  claim 1 , wherein the second nucleotide sequence encodes the human β 2  adrenergic receptor protein, into the bacterial cell; and  
 b) culturing the bacterial cell under conditions whereby the second nucleotide sequence of the nucleic acid is expressed to produce the human β 2  adrenergic receptor protein.  
 
     
     
         33 . A method of producing a rat renal outer medullary K +  channel protein in a bacterial cell comprising: 
 a) introducing the nucleic acid of  claim 1 , wherein the second nucleotide sequence encodes the rat renal outer medullary K +  channel protein, into the bacterial cell; and  
 b) culturing the bacterial cell under conditions whereby the second nucleotide sequence of the nucleic acid is expressed to produce the rat renal outer medullary K +  channel protein.  
 
     
     
         34 . A method of producing a human small G-protein rho protein in a bacterial cell comprising: 
 a) introducing the nucleic acid of  claim 1 , wherein the second nucleotide sequence encodes the small G-protein rho protein, into the bacterial cell; and    b) culturing the bacterial cell under conditions whereby the second nucleotide sequence of the nucleic acid is expressed to produce the small G-protein rho protein.