US2014081003A1PendingUtilityA1

Methods and compositions for preventing norleucine misincorporation into proteins

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Assignee: GENENTECH INCPriority: Sep 19, 2012Filed: Sep 19, 2013Published: Mar 20, 2014
Est. expirySep 19, 2032(~6.1 yrs left)· nominal 20-yr term from priority
Inventors:Michael W. LairdKarthik Veeravalli
C12N 9/1029C07K 16/22C07K 2317/14C12N 9/1085C07K 16/18C12N 15/70C12Y 203/01046C07K 16/2863C07K 16/32C12P 21/00C07K 16/40C12Y 205/01006C12P 21/02
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Claims

Abstract

The present invention relates to methods and compositions for preventing incorporation of norleucine into proteins during recombinant protein production in bacteria. The present invention also provides microorganism host cells and nucleic acid molecules for use with the methods and compositions provided herein.

Claims

exact text as granted — not AI-modified
What is claimed is: 
     
         1 . A method for preventing or reducing norleucine misincorporation into a protein or polypeptide, the method comprising expressing the protein or the polypeptide in a microorganism, wherein the microorganism is a mutant microorganism which produces methionine to a degree or extent sufficient to prevent or reduce norleucine misincorporation into the protein or polypeptide. 
     
     
         2 . The method of  claim 1 , wherein the microorganism is a bacteria. 
     
     
         3 . The method of  claim 1 , wherein the microorganism is  E. coli.    
     
     
         4 . The method of  claim 1 , wherein the microorganism is a feedback-resistant or feedback-insensitive homoserine succinyltransferase microorganism. 
     
     
         5 . The method of  claim 1 , wherein the microorganism is de-repressed for methionine production. 
     
     
         6 . The method of  claim 1 , wherein the microorganism comprises a mutant metA allele, a mutant metK allele, or a mutant metA allele and a mutant metK allele. 
     
     
         7 . The method of  claim 1 , wherein expressing the protein or the polypeptide in the microorganism is performed in the absence of exogenously added methionine to the culture medium or without a methionine feed. 
     
     
         8 . A microorganism comprising a mutant metA allele, a mutant metK allele, or a mutant metA allele and a mutant metK allele. 
     
     
         9 . The microorganism of  claim 8 , wherein the microorganism comprises a mutant metA allele, wherein the mutant metA allele comprises a nucleic acid sequence encoding an amino acid substitution in MetA selected from the group consisting of an arginine to cysteine substitution at amino acid position 27, a glutamine to glutamic acid substitution at amino acid position 64, a tyrosine to cysteine substitution at amino acid position 294, an isoleucine to serine substitution at amino acid position 296, a proline to leucine substitution at amino acid position 298, and an isoleucine to serine substitution at amino acid position 296 and a proline to leucine substitution at amino acid position 298. 
     
     
         10 . The microorganism of  claim 9 , wherein the microorganism comprises a mutant metA allele, wherein the mutant metA allele comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26. 
     
     
         11 . The microorganism of  claim 8 , wherein the mutant metK allele comprises a nucleic acid sequence encoding an amino acid substitution in MetK comprising a valine to glutamic acid substitution at amino acid position 185 or a nucleic acid sequence comprising a deletion of the cytosine base at nucleic acid residue position 1132 of the metK allele. 
     
     
         12 . The microorganism of  claim 11 , wherein the microorganism comprises a mutant metK allele, wherein the mutant metK allele comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO:27 and SEQ ID NO:28. 
     
     
         13 . The microorganism of  claim 8 , wherein the microorganism comprises a mutant metA allele, wherein the mutant metA allele comprises a nucleic acid sequence encoding an amino acid substitution in MetA selected from the group consisting of an arginine to cysteine substitution at amino acid position 27, a glutamine to glutamic acid substitution at amino acid position 64, a tyrosine to cysteine substitution at amino acid position 294, an isoleucine to serine substitution at amino acid position 296, a proline to leucine substitution at amino acid position 298, and an isoleucine to serine substitution at amino acid position 296 and a proline to leucine substitution at amino acid position 298, and further wherein the mutant metK allele comprises a nucleic acid sequence encoding an amino acid substitution in MetK comprising a valine to glutamic acid substitution at amino acid position 185 or a nucleic acid sequence comprising a deletion of the cytosine base at nucleic acid residue position 1132 of the metK allele. 
     
     
         14 . The microorganism of  claim 13 , wherein the microorganism comprises a mutant metA allele, wherein the mutant metA allele comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26, and further wherein the microorganism comprises a mutant metK allele, wherein the mutant metK allele comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO:27 and SEQ ID NO:28. 
     
     
         15 . The microorganism of  claim 8 , further comprising nucleic acid encoding an anti-VEGF antibody or an anti-VEGF antibody fragment. 
     
     
         16 . The microorganism of  claim 15 , wherein nucleic acid encoding an anti-VEGF antibody or an anti-VEGF antibody fragment is nucleic acid encoding the amino acid sequence of SEQ ID NO:46 and nucleic acid encoding the amino acid sequence of SEQ ID NO:47. 
     
     
         17 . The microorganism of  claim 15 , wherein nucleic acid encoding an anti-VEGF antibody of an anti-VEGF antibody fragment is selected from the group consisting of the nucleic acid sequence comprising SEQ ID NO: 33 and the nucleic acid sequence comprising SEQ ID NO:34. 
     
     
         18 . The microorganism of  claim 8 , further comprising nucleic acid encoding an anti-Factor D antibody or an anti-Factor D antibody fragment. 
     
     
         19 . The microorganism of  claim 18 , wherein nucleic acid encoding an anti-Factor D antibody or an anti-Factor D antibody fragment is nucleic acid encoding the amino acid sequence of SEQ ID NO:48 and nucleic acid encoding the amino acid sequence of SEQ ID NO:49. 
     
     
         20 . The microorganism of  claim 8 , further comprising nucleic acid encoding an anti-MET antibody or an anti-MET antibody fragment. 
     
     
         21 . The microorganism of  claim 20 , wherein nucleic acid encoding an anti-MET antibody or an anti-MET antibody fragment is selected from the group consisting of nucleic acid encoding the amino acid sequence of SEQ ID NO:50, nucleic acid encoding the amino acid sequence of SEQ ID NO:51, and nucleic acid encoding the amino acid sequence of SEQ ID NO:52. 
     
     
         22 . A method for producing a protein or a polypeptide in a bacteria host cell, wherein the protein or the polypeptide is free of norleucine misincorporation, the method comprising expressing in the bacteria host cell a nucleic acid encoding the protein or the polypeptide under culture conditions suitable to allow for expression of the protein or the polypeptide, wherein the bacteria host cell comprises a mutant metA allele, a mutant metK allele, or a mutant metA allele and a mutant metK allele, thereby producing a protein or a polypeptide free of norleucine misincorporation. 
     
     
         23 . The method of  claim 22 , wherein the bacteria host cell is selected from the group consisting of the microorganism of  claim 9 , the microorganism of  claim 10 , the microorganism of  claim 11 , the microorganism of  claim 12 , the microorganism of  claim 13 , and the microorganism of  claim 14 . 
     
     
         24 . The method of  claim 22 , wherein the protein or the polypeptide is an antibody or an antibody fragment. 
     
     
         25 . The method of  claim 23 , wherein the protein or the polypeptide is an antibody or an antibody fragment. 
     
     
         26 . The method of  claim 24 , wherein the antibody or the antibody fragment is an anti-VEGF antibody or an anti-VEGF antibody fragment. 
     
     
         27 . The method of  claim 26 , wherein nucleic acid encoding the anti-VEGF antibody or the anti-VEGF antibody fragment is nucleic acid encoding the amino acid sequence of SEQ ID NO:46 and nucleic acid encoding the amino acid sequence of SEQ ID NO:47. 
     
     
         28 . The method of  claim 26 , wherein nucleic acid encoding the anti-VEGF antibody or the anti-VEGF antibody fragment is selected from the group consisting of the nucleic acid sequence comprising SEQ ID NO: 33 and the nucleic acid sequence comprising SEQ ID NO:34. 
     
     
         29 . The method of  claim 24 , wherein the antibody or the antibody fragment is an anti-Factor D antibody or an anti-Factor D antibody fragment. 
     
     
         30 . The method of  claim 29 , wherein nucleic acid encoding the anti-Factor D antibody or the anti-Factor D antibody fragment is nucleic acid encoding the amino acid sequence of SEQ ID NO:48 and nucleic acid encoding the amino acid sequence of SEQ ID NO:49. 
     
     
         31 . The method of  claim 24 , wherein the antibody or the antibody fragment is an anti-MET antibody or an anti-MET antibody fragment. 
     
     
         32 . The method of  claim 31 , wherein nucleic acid encoding an anti-MET antibody or an anti-MET antibody fragment is selected from the group consisting of nucleic acid encoding the amino acid sequence of SEQ ID NO:50, nucleic acid encoding the amino acid sequence of SEQ ID NO:51, and nucleic acid encoding the amino acid sequence of SEQ ID NO:52. 
     
     
         33 . The method of  claim 22 , wherein expressing the protein or the polypeptide in the bacteria host cell is performed in the absence of exogenously added methionine to the culture medium or without a methionine feed. 
     
     
         34 . An anti-VEGF antibody or anti-VEGF antibody fragment produced according to the method of  claim 26 ,  claim 27 , or  claim 28 . 
     
     
         35 . An anti-Factor D antibody or anti-Factor D antibody fragment produced according to the method of  claim 29  or  claim 30 . 
     
     
         36 . An anti-MET antibody or anti-MET antibody fragment produced according to the method of  claim 31  or  claim 32 .

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