Method of expressing recombinant protein in cho cells

Abstract

Method of expressing recombinant protein in CHO cells, by using an expression vector comprising the murine IgG 2A gene locus.

1 - 8 . (canceled)  
     
     
         9 . CHO cell transfected with an expression vector comprising a promoter that is active in CHO cells and that is driving expression of a recombinant product protein and further comprising a portion from the murine IgA 2A gene locus DNA which portion is enhancing activity of said promoter.  
     
     
         10 . CHO cell according to  claim 9 , characterized in that the vector further comprises a transcription unit encoding a selectable marker, preferably a glutamin synthetase (GS) marker.  
     
     
         11 . CHO cell according to  claim 9  or  10 , characterized in the CHO cell is stably transfected.  
     
     
         12 . Method of expressing a recombinant protein, comprising the steps of culturing a CHO cell transfected with an expression vector comprising a promoter active in CHO cells driving expression of a recombinant product protein and further comprising the murine IgA 2A gene locus DNA or a DNA sequence variant or DNA fragment thereof which is enhancing activity of said promoter, and harvesting the product protein  
     
     
         13 . Method according to  claim 12 , characterised in that the promoter is a strong viral promoter, preferably the hCMV promoter.  
     
     
         14 . Method according to one of claims  12  or  13 , characterised in that the IgA 2A gene locus portion does lack the natural immunoglobulin promoter.  
     
     
         15 . Method according to  claim 12 , characterized in that the promoter is hCMV promoter or a functional part thereof having promoter activity wherein said promoter or functional part lack the ‘modulator’ sequence in the upstream/enhancer portion as found stretching from position −750 to −1150 relative to the MIE transcription start site.  
     
     
         16 . CHO cell transfected with a mammalian expression vector comprising at least a first transcription unit for a product gene which transcription unit is under the control of the mCMV promoter, and further comprising a second transcription unit comprising a glutamine synthetase (GS) marker gene.  
     
     
         17 . Mammalian expression vector comprising at least a first transcription unit for a product gene which transcription unit is under control of the mCMV promoter or a functional fragment thereof, and further comprising a second transcription unit comprising a glutamine synthetase (GS) marker gene.  
     
     
         18 . Vector according to  claim 16 , wherein the mCMV promoter or functional fragment comprises the natural transcription start site (+0) and extends upstream to position −500.  
     
     
         19 . Vector according to  claim 18 , wherein the mCMV promoter or functional fragment extends to the natural Xho l restriction site.  
     
     
         20 . Vector according to  claim 18 , wherein the transcription start site is engineered to comprise a suitable restriction site for insertion of a recombinant gene product.  
     
     
         21 . Vector according to  claim 17  or  18 , wherein the first transcription unit harbors at least one intron sequence.  
     
     
         22 . Vector according to  claim 21 , wherein said intron is not the first, natural intron of the mCMV promoter.  
     
     
         23 . Method of using  17  for enhancing the transfection rate in CHO cells.