Recombinant protein production in heterologous systems

Abstract

Isolated polynucleotides are disclosed which increase the efficiency of gene expression in a heterologous cell. The polynucleotide sequences which encode polypeptides are adapted such that the average rate of translation of the first at least about 30 amino acids is slower by at least two fold than the average rate of translation of the remaining amino acids of the polypeptide.

What is claimed is: 
     
         1 . A method of increasing the efficiency of gene expression in a non-human cell, the method comprising adapting a sequence of a polynucleotide which encodes a human polypeptide having a predetermined amino acid sequence, wherein the codon usage at the 5′ end of the polynucleotide is modified such that the average rate of translation of the first 30 amino acids is slower by no more than 2.5 fold and no less than 1.3 fold than the average rate of translation of the remaining amino acids of the polypeptide in said non-human cell, wherein said codon usage does not alter the predetermined amino acid sequence, wherein at least 60% of the codons for said first 30 amino acids correspond to low abundance tRNAs in said non-human cell, thereby increasing the efficiency of gene expression in the non-human cell. 
     
     
         2 . The method of  claim 1 , wherein the 3′ end of said polynucleotide is non-modified. 
     
     
         3 . The method of  claim 1 , wherein the 3′ end of said polynucleotide is modified. 
     
     
         4 . The method of  claim 1 , wherein said non-human cells comprise bacterial cells, a fungal cells, plant cells, insect cells and mammalian cells. 
     
     
         5 . The method of  claim 1 , wherein said non-human cells are selected from the group consisting of  E. Coli  cells, Chinese hamster ovary (CHO) cells and  S. cerevisiae  cells. 
     
     
         6 . The method of  claim 1 , wherein said non-human cells comprise Chinese hamster ovary (CHO) cells. 
     
     
         7 . The method of  claim 1 , wherein said polypeptide is selected from the group consisting of an antibody, insulin, interferon, growth hormone, erythropoietin, growth hormone, follicle stimulating hormone, factor VIII, low density lipoprotein receptor (LDLR) alpha galactosidase A and glucocerebrosidase. 
     
     
         8 . The method of  claim 1 , wherein said polypeptide comprises a signal sequence. 
     
     
         9 . The method of  claim 1 , wherein said polynucleotide comprises a sequence as set forth in SEQ ID NOs: 2-7. 
     
     
         10 . A method of generating a polypeptide, the method comprising:
 (a) inserting an isolated polynucleotide into a cell, said cell being of said another species, wherein said isolated polynucleotide encodes a human polypeptide having a predetermined amino acid sequence, wherein the codon usage at the 5′ end of the polynucleotide is modified such that the average rate of translation of the first 30 amino acids is slower by no more than 2.5 fold and no less than 1.3 fold than the average rate of translation of the remaining amino acids of the polypeptide in non-human cells, wherein said codon usage does not alter the predetermined amino acid sequence, wherein at least 60% of the codons for said first 30 amino acids correspond to low abundance tRNAs in said non-human cells; and   (b) culturing the cell under conditions that allows expression of the polypeptide, thereby generating the polypeptide.   
     
     
         11 . The method of  claim 10 , further comprising purifying the polypeptide following said culturing. 
     
     
         12 . The method of  claim 10 , wherein the 3′ end of said polynucleotide is non-modified. 
     
     
         13 . The method of  claim 10 , wherein the 3′ end of said polynucleotide is modified. 
     
     
         14 . The method of  claim 10 , wherein said non-human cells comprise bacterial cells, fungal cells, plant cells, insect cells and mammalian cells. 
     
     
         15 . The method of  claim 10 , wherein said non-human cells are selected from the group consisting of  E. Coli  cells, Chinese hamster ovary (CHO) cells and  S. cerevisiae  cells. 
     
     
         16 . The method of  claim 10 , wherein said non-human cells comprise Chinese hamster ovary (CHO) cells. 
     
     
         17 . The method of  claim 10 , wherein said polypeptide is selected from the group consisting of an antibody, insulin, interferon, growth hormone, erythropoietin, growth hormone, follicle stimulating hormone, factor VIII, low density lipoprotein receptor (LDLR) alpha galactosidase A and glucocerebrosidase. 
     
     
         18 . The method of  claim 10 , wherein said polypeptide comprises a signal sequence. 
     
     
         19 . The method of  claim 10 , wherein said polynucleotide comprises a sequence as set forth in SEQ ID NOs: 2-7.