Synthetic genes for enhanced expression

Abstract

A synthetic nucleic acid sequence is disclosed, comprising a non-naturally occurring polymer of nucleic acids, having a biological function encoded by the sequence and known from a starting nucleic acid sequence, and having a difference in sequence of at least about 5% between the nucleic acids of the synthetic sequence and the starting sequence. The difference between the nucleic acid sequences results in a different free energy of folding for the synthetic sequence as compared to the starting sequence, such that the synthetic sequence would be expressed better in a selected heterologous host cell than the starting sequence would be if expressed in the same heterologous host cell.

1 - 49 . (canceled)  
     
     
         50 . A method of designing a synthetic polynucleotide, the method comprising: 
 providing a starting polynucleotide;    determining the predicted free energy of folding per base of the starting polynucleotide;    modifying the starting polynucleotide by replacing at least one codon from the starting polynucleotide with a different codon to provide a modified polynucleotide;    determining the predicted free energy of folding per base of the modified polynucleotide; and    comparing the predicted free energy of folding of the modified polynucleotide with the predicted free energy of folding of the starting polynucleotide.    
     
     
         51 . The method of  claim 50  wherein the different codon corresponds to the same amino acid as the replaced codon.  
     
     
         52 . The method of  claim 50  further comprising: 
 (a) determining whether the predicted free energy of folding of the modified polynucleotide is changed relative to the predicted free energy of folding of the starting polynucleotide by a desired amount; and    (b) if the predicted free energy of folding of the modified polynucleotide is not changed by the desired amount, further modifying the modified polynucleotide by replacing at least one codon from the modified polynucleotide with a different corresponding codon to provide a different modified polynucleotide.    
     
     
         53 . The method of  claim 52  further comprising repeating steps (a) and (b) until the predicted free energy of folding of the modified polynucleotide is increased by the desired amount to ultimately provide a final polynucleotide.  
     
     
         54 . The method of  claim 52  wherein the desired free energy of folding of the modified polynucleotide is increased as compared to the predicted free energy of folding of the starting polynucleotide.  
     
     
         55 . The method of  claim 50  comprising physically creating the modified polynucleotide.  
     
     
         56 . The method of  claim 52  comprising physically creating the different modified polynucleotide.  
     
     
         57 . The method of  claim 53  comprising physically creating the final polynucleotide.  
     
     
         58 . A synthetic polynucleotide designed by the method of  claim 50 .  
     
     
         59 . A synthetic polynucleotide designed by the method of  claim 52 .  
     
     
         60 . A synthetic polynucleotide designed by the method of  claim 53 .  
     
     
         61 . A nucleic acid having one of the sequence of a polynucleotide designed by the method of  claim 50  and a sequence complementary thereto.  
     
     
         62 . A nucleic acid having one of the sequence of a polynucleotide designed by the method of  claim 52  and a sequence complementary thereto.  
     
     
         63 . A nucleic acid having one of the sequence of a polynucleotide designed by the method of  claim 53  and a sequence complementary thereto.  
     
     
         64 . The method of  claim 50  wherein the codon replaced to make the modified polynucleotide corresponds to a different amino acid than was replaced in the starting polynucleotide.  
     
     
         65 . The method of  claim 50  wherein the different codon is selected from the most frequently used by a selected host.  
     
     
         66 . The method of  claim 52  wherein the starting polynucleotide is derived from a eukaryotic cell and is modified to be expressed in a selected prokaryotic host cell.  
     
     
         67 . A method of physically creating a tangible synthetic polynucleotide comprising creating a physical embodiment of the synthetic polynucleotide of  claim 58 .  
     
     
         68 . A method of physically creating a tangible synthetic polynucleotide comprising creating a physical embodiment of the synthetic polynucleotide of  claim 59 .  
     
     
         69 . A method of physically creating a tangible synthetic polynucleotide comprising creating a physical embodiment of the synthetic polynucleotide of  claim 60 .  
     
     
         70 . A physical embodiment of the tangible synthetic polynucleotide prepared by the method of  claim 67 .  
     
     
         71 . A physical embodiment of the tangible synthetic polynucleotide prepared by the method of  claim 68 .  
     
     
         72 . A physical embodiment of the tangible synthetic polynucleotide prepared by the method of  claim 69.