Methods of modulating nucleic acid stability and protein expression

Abstract

A synthetic nucleic acid which encodes a protein wherein at least one optimal or non-optimal codon in a wild type nucleic acid encoding the protein has been replaced respectively with one or more non-optimal codons or optimal codons encoding the same amino acid.

1 - 22 . (canceled) 
     
     
         23 . A method for preparing a synthetic nucleic acid encoding a protein expressed by a eukaryotic cell, comprising:
 identifying optimal and non-optimal codons in a nucleic acid encoding the protein, and   replacing one or more of the optimal codons with a non-optimal codon encoding the same amino acid as the replaced codon or replacing one or more of the non-optimal codons with an optimal codon encoding the same amino acid, wherein the replacement of the one or more codons from the nucleic acid encoding the protein modulates expression of the protein in the eukaryotic cell at least about 10% different compared to that expressed by the nucleic acid prior to replacement in an in vitro mammalian cell culture system under identical conditions, and wherein the optimal codons are selected from the group consisting of get (Alanine), ggt (Glycine), gtc (Valine), ttg (Leucine), gtt (Valine), gcc (Alanine), cca (Proline), act (Threonine), tct (Serine), tcc (Serine), gaa (Glutamaic Acid), cgt (Arginine), caa (Glutamine), att (Isoleucine), aga (Arginine), and tgt (Cysteine); and the non-optimal codons are selected from the group consisting of ggc (Glycine), tgg (Tryptophan), atg (Methionine), tgc (Cysteine), ccc (Proline), gtg (Valine), cgc (Arginine), gag (Glutamaic Acid), cag (Glutamine), agc (Serine), and ctg (Leucine).   
     
     
         24 . The method of  claim 23 , wherein the synthetic nucleic sequence is capable of expressing the protein at a level which is at least 50% different compared to that expressed by the nucleic acid prior to replacement in an in vitro mammalian cell culture system under identical conditions. 
     
     
         25 . The method of  claim 23 , wherein the synthetic nucleic sequence is capable of expressing the protein at a level which is at least 75% different compared to that expressed by the nucleic acid prior to replacement in an in vitro mammalian cell culture system under identical conditions. 
     
     
         26 . The method of  claim 23 , wherein one or more of the optimal codons is replaced with a non-optimal codon encoding the same amino acid as the replaced codon so that the synthetic nucleic acid sequence has less than about 40% optimal codons. 
     
     
         27 . The method of  claim 23 , wherein one or more of the non-optimal codons is replaced with an optimal codon encoding the same amino acid as the replaced codon so that the synthetic nucleic acid sequence has more than about 70% optimal codons. 
     
     
         28 . The method of  claim 23 , wherein the nucleic acid prior to replacement comprising a wild type nucleic acid. 
     
     
         29 . The method of  claim 23 , the eukaryotic cell is a yeast cell. 
     
     
         30 . The method of  claim 23 , wherein the eukaryotic cell is a mammalian cell. 
     
     
         31 . The method of  claim 30 , wherein the mammalian cell is a COS (Chinese Hamster Ovary) cell. 
     
     
         32 . The method of  claim 23 , wherein the eukaryotic cell is a plant cell. 
     
     
         33 . The method of  claim 23 , wherein the synthetic nucleic acid comprises in vitro transcribed mRNA. 
     
     
         34 . The method of  claim 23 , wherein the synthetic nucleic comprises DNA. 
     
     
         35 . A method for producing a recombinant protein comprising: providing a synthetic nucleic acid sequence of  claim 23 ; ligating the synthetic nucleic acid sequence into an expression vector; transfecting the host cell with the expression vector; culturing the transfected host cell in a suitable culture media appropriate for the expression of the protein and isolating the protein. 
     
     
         36 . The method of  claim 35 , wherein the synthetic nucleic sequence is capable of expressing the protein at a level which is at least 50% different compared to that expressed by the nucleic acid prior to replacement in an in vitro mammalian cell culture system under identical conditions. 
     
     
         37 . The method of  claim 35 , wherein the synthetic nucleic sequence is capable of expressing the protein at a level which is at least 75% different compared to that expressed by the nucleic acid prior to replacement in an in vitro mammalian cell culture system under identical conditions. 
     
     
         38 . The method of  claim 35 , wherein one or more of the optimal codons is replaced with a non-optimal codon encoding the same amino acid as the replaced codon so that the synthetic nucleic acid sequence has less than about 40% optimal codons. 
     
     
         39 . The method of  claim 35 , wherein one or more of the non-optimal codons is replaced with an optimal codon encoding the same amino acid as the replaced codon so that the synthetic nucleic acid sequence has more than about 70% optimal codons. 
     
     
         40 . The method of  claim 35 , wherein the nucleic acid prior to replacement comprising a wild type nucleic acid. 
     
     
         41 . The method of  claim 35 , wherein the host cell is a eukaryotic cell. 
     
     
         42 . The method of  claim 41 , the eukaryotic cell is a yeast cell. 
     
     
         43 - 56 . (canceled)