Cmv promoter and method for production of polypeptides

Abstract

The current invention reports a promoter that has the nucleic acid sequence of SEQ ID NO: 02 or SEQ ID NO: 03 which is a human CMV major immediate-early (hCMV-MIE) promoter/enhancer with C to G point mutation at position −41 and/or −179 relative to the transcription start site. This new promoter is especially useful for the production of polypeptides at large scale as it shows reduced promoter silencing and improved polypeptide production.

1 . A human CMV promoter that has a nucleotide G at a nucleotide position −41 and/or at position −179 relative to a transcription start site. 
     
     
         2 . A promoter that has the nucleic acid sequence of SEQ ID NO: 02 or SEQ ID NO: 03. 
     
     
         3 . A nucleic acid consisting of the nucleic acid of SEQ ID NO: 02 or SEQ ID NO: 03, and wherein the nucleic acid has a promoter strength of at least 80% of the human CMV-major immediate-early promoter of SEQ ID NO: 01 when the nucleic acid is operably linked to a nucleic acid of SEQ ID NO: 04. 
     
     
         4 . A method for the production of a polypeptide, said method comprising the following steps:
 a) transfecting a eukaryotic cell with a nucleic acid comprising an expression cassette comprising a first nucleic acid of SEQ ID NO: 02 or SEQ ID NO: 03 operably linked to a second nucleic acid encoding the polypeptide,   b) selecting a cell transfected in step a),   c) cultivating the selected cell of step b),   d) recovering the polypeptide from the cell or the cultivation medium, and thereby producing the polypeptide.   
     
     
         5 . The method according to  claim 4  wherein the production is a production at large scale. 
     
     
         6 . The method according to  claim 4  wherein the polypeptide is an immunoglobulin light chain or an immunoglobulin heavy chain or a variant thereof, or a fragment thereof or a fusion thereof. 
     
     
         7 . The method according to  claim 4  wherein the nucleic acid comprises a further expression cassette encoding a selectable marker. 
     
     
         8 . The method according to  claim 4  wherein said eukaryotic cell is a mammalian cell. 
     
     
         9 . The method according to  claim 8 , wherein said mammalian cell is a CHO cell, a BHK cell, a HEK cell, a Sp2/0 cell or a Per.C6® cell. 
     
     
         10 . The method according to  claim 9 , wherein said mammalian cell is a CHO cell or a HEK cell. 
     
     
         11 . The method according to  claim 4  wherein said polypeptide is an immunoglobulin, or an immunoglobulin-fragment, or an immunoglobulin-conjugate. 
     
     
         12 . The method according to  claim 8  wherein said selectable marker is a dihydrofolate reductase, an aminoglycoside phosphotransferase or a hygromycin-phosphotransferase. 
     
     
         13 . A cell comprising the promoter according to  claim 1 .