US2012301955A1PendingUtilityA1

Modified messenger RNA stabilizing sequences for expressing genes in bacterial cells

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Assignee: THOMAS MICHAELPriority: Dec 21, 2006Filed: Aug 13, 2012Published: Nov 29, 2012
Est. expiryDec 21, 2026(~0.4 yrs left)· nominal 20-yr term from priority
Inventors:Michael D. ThomasGloria ErichsenWilliam Widner
C07K 14/32C12N 15/75
46
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Claims

Abstract

The present invention relates to methods of producing a polypeptide having biological activity in a bacterial cell, comprising: (a) cultivating a bacterial host cell in a medium conducive for production of the polypeptide, wherein the bacterial host cell comprises a nucleic acid construct comprising a promoter region operably linked to a polynucleotide sequence encoding the polypeptide and a modified mRNA processing/stabilizing sequence located downstream of the promoter region and upstream of the ribosome binding site of the polynucleotide sequence encoding the polypeptide, wherein the modified mRNA processing/stabilizing sequence promotes higher expression of the polynucleotide sequence compared to an unmodified mRNA processing/stabilizing sequence; and (b) isolating the polypeptide having biological activity from the cultivation medium. The present invention also relates to such modified mRNA processing/stabilizing sequences, nucleic acid constructs, and bacterial host cells and to methods of obtaining such bacterial host cells.

Claims

exact text as granted — not AI-modified
1 . An isolated nucleic acid comprising a modified mRNA processing/stabilizing sequence, wherein the modified mRNA processing/stabilizing sequence comprises at least one additional copy of a Shine-Dalgarno sequence. 
     
     
         2 . The isolated nucleic acid of  claim 1 , wherein the modified mRNA processing/stabilizing sequence is a cryIIIA mRNA processing/stabilizing sequence comprising at least one additional copy of a Shine-Dalgarno sequence. 
     
     
         3 . The isolated nucleic acid of  claim 1 , wherein the modified mRNA processing/stabilizing sequence is the cryIIIA mRNA processing/stabilizing sequence of SEQ ID NO: 4 comprising at least one additional copy of a Shine-Dalgarno sequence. 
     
     
         4 . The isolated nucleic acid of  claim 1 , wherein the modified mRNA processing/stabilizing sequence is a SP82 mRNA processing/stabilizing sequence comprising at least one additional copy of a Shine-Dalgarno sequence. 
     
     
         5 . The isolated nucleic acid of  claim 1 , wherein the modified mRNA processing/stabilizing sequence is the SP82 mRNA processing/stabilizing sequence of SEQ ID NO: 5 comprising at least one additional copy of a Shine-Dalgarno sequence. 
     
     
         6 . The isolated nucleic acid of  claim 1 , wherein the at least one additional copy of a Shine-Dalgarno sequence comprises the sequence GGAG. 
     
     
         7 . The isolated nucleic acid of  claim 1 , wherein the at least one additional copy of a Shine-Dalgarno sequence comprises the sequence GGAGG. 
     
     
         8 . The isolated nucleic acid of  claim 1 , wherein the at least one additional copy of a Shine-Dalgarno sequence comprises SEQ ID NO: 1. 
     
     
         9 . The isolated nucleic acid of  claim 1 , wherein the modified mRNA processing/stabilizing sequence comprises at least two additional copies of a Shine-Dalgarno sequence. 
     
     
         10 . A nucleic acid construct comprising a promoter region operably linked to the nucleic acid of  claim 1  and a polynucleotide encoding a polypeptide having biological activity, wherein the nucleic acid of  claim 1  is located downstream of the promoter region and upstream of the ribosome binding site of the polynucleotide encoding the polypeptide having biological activity. 
     
     
         11 . The nucleic acid construct of  claim 10 , wherein the modified mRNA processing/stabilizing sequence of the nucleic acid of  claim 1  promotes higher expression of the polynucleotide encoding a polypeptide having biological activity compared to an unmodified mRNA processing/stabilizing sequence. 
     
     
         12 . The nucleic acid construct of  claim 10 , wherein the modified mRNA processing/stabilizing sequence is a cryIIIA mRNA processing/stabilizing sequence comprising at least one additional copy of a Shine-Dalgarno sequence. 
     
     
         13 . The nucleic acid construct of  claim 10 , wherein the modified mRNA processing/stabilizing sequence is the cryIIIA mRNA processing/stabilizing sequence of SEQ ID NO: 4 comprising at least one additional copy of a Shine-Dalgarno sequence. 
     
     
         14 . The nucleic acid construct of  claim 10 , wherein the modified mRNA processing/stabilizing sequence is a SP82 mRNA processing/stabilizing sequence comprising at least one additional copy of a Shine-Dalgarno sequence. 
     
     
         15 . The nucleic acid construct of  claim 10 , wherein the modified mRNA processing/stabilizing sequence is the SP82 mRNA processing/stabilizing sequence of SEQ ID NO: 5 comprising at least one additional copy of a Shine-Dalgarno sequence. 
     
     
         16 . The nucleic acid construct of  claim 10 , wherein the at least one additional copy of a Shine-Dalgarno sequence comprises the sequence GGAG. 
     
     
         17 . The nucleic acid construct of  claim 10 , wherein the at least one additional copy of a Shine-Dalgarno sequence comprises the sequence GGAGG. 
     
     
         18 . The nucleic acid construct of  claim 10 , wherein the at least one additional copy of a Shine-Dalgarno sequence comprises SEQ ID NO: 1. 
     
     
         19 . The nucleic acid construct of  claim 10 , wherein the modified mRNA processing/stabilizing sequence comprises at least two additional copies of a Shine-Dalgarno sequence. 
     
     
         20 . A recombinant expression vector comprising the nucleic acid construct of  claim 10 .

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