Method for the expression of polypeptides using modified nucleic acids

Abstract

Herein is reported a method for recombinantly producing a polypeptide in a cell comprising the step of cultivating a cell which comprises a nucleic acid encoding the polypeptide, and recovering the polypeptide from the cell or the cultivation medium, wherein each of the amino acid residues of the polypeptide is encoded by at least one codon, whereby the different codons encoding the same amino acid residue are combined in one group and each of the codons in a group is defined by a specific usage frequency within the group, whereby the sum of the specific usage frequencies of all codons in one group is 100%, and wherein the usage frequency of a codon in the polypeptide encoding nucleic acid is about the same as its specific usage frequency within its group.

1 . A method for recombinantly producing a polypeptide in a prokaryotic cell comprising the step of cultivating a prokaryotic cell which comprises a nucleic acid encoding the polypeptide, and recovering the polypeptide from the prokaryotic cell or the cultivation medium,
 wherein each of the amino acid residues of the polypeptide is encoded by at least one codon, whereby the codon(s) encoding the same amino acid residue are combined in one group and each of the codons in a group is defined by a specific usage frequency within the group, whereby the sum of the specific usage frequencies of all codons in one group is 100%,   wherein the overall usage frequency of each codon in the polypeptide encoding nucleic acid is about the same as its specific usage frequency within its group.   
     
     
         2 . The method according to  claim 1 , characterized in that the groups comprises only codons with an overall usage frequency within the genome of the cell of more than 5%. 
     
     
         3 . The method according to  claim 1 , characterized in that the groups comprises only codons with an overall usage frequency within the genome of the cell of 8% or more. 
     
     
         4 . The method according to  claim 1 , characterized in that the groups comprises only codons with an overall usage frequency within the genome of the cell of 10% or more. 
     
     
         5 . The method according to  claim 1 , characterized in that for each sequential occurrence of a specific amino acid in the polypeptide starting from the N-terminus of the polypeptide in the corresponding position of the encoding nucleic acid the same codon is used as that which is present at the corresponding sequential position in the respective amino acid codon motif for the specific amino acid. 
     
     
         6 . The method according to  claim 5 , characterized in that
 i) after usage of the final codon of the amino acid codon motif at the next occurrence of the specific amino acid in the polypeptide the codon that is at the first position of the respective amino acid codon motif is used again in the corresponding encoding nucleic acid,   ii) for each further sequential occurrence of this specific amino acid in the polypeptide in the corresponding position of the encoding nucleic acid the codon is used which is present at the corresponding position in the respective amino acid codon motif for the specific amino acid.   
     
     
         7 . The method according to  claim 5 , characterized in that the codons in the amino acid codon motif are arranged with decreasing specific usage frequency whereby after the codon with the lowest specific usage frequency or the codon with the second lowest specific usage frequency the codon with the highest specific usage frequency is used. 
     
     
         8 . The method according to  claim 7 , characterized in that the codons in the amino acid codon motif are arranged with decreasing specific usage frequency whereby after the codon with the lowest specific usage frequency the codon with the highest specific usage frequency is used. 
     
     
         9 . The method according to  claim 1 , characterized in that the cell is  E. coli.    
     
     
         10 . The method according to  claim 9 , characterized in that the amino acid codon motif for
 alanine is selected from SEQ ID NO: 01, 02, 03, 04 and 05, and/or   arginine is selected from SEQ ID NO: 06 and 07, and/or   asparagine is selected from SEQ ID NO: 08, 09, 10, 11, and 12, and/or   aspartic acid is selected from SEQ ID NO: 13 and 14, and/or,   cysteine is selected from SEQ ID NO: 15, 16 and 17, and/or   glutamine is selected from SEQ ID NO: 18, 19, 20, and 21, and/or   glutamic acid is selected from SEQ ID NO: 22, 23 and 24, and/or   glycine is selected from SEQ ID NO: 25 and 26, and/or   histidine is selected from SEQ ID NO: 27 and 28, and/or   isoleucine is selected from SEQ ID NO: 29 and 30, and/or   leucine is selected from SEQ ID NO: 31, 32 and 33, and/or   lysine is selected from SEQ ID NO: 34, 35, 36 and 37, and/or   phenylalanine is selected from SEQ ID NO: 38, 39 and 40, and/or   proline is selected from SEQ ID NO: 41, 42, 43, 44, 45 and 46, and/or   serine is selected from, SEQ ID NO: 47 and 48, and/or   threonine is selected from SEQ ID NO: 49, 50 and 51, and/or   tyrosine is selected from SEQ ID NO: 52 and 53, and/or   valine is selected from SEQ ID NO: 54, 55 and 56.   
     
     
         11 . The method according to any one of  claims 1  to  10 , characterized in that the polypeptide is an antibody, or an antibody fragment, or an antibody fusion polypeptide.