Selection of host cells expressing protein at high levels

Abstract

The invention provides a DNA molecule comprising an open reading frame sequence that encodes a selectable marker polypeptide, wherein said DNA molecule in the coding strand comprises a translation start sequence for the selectable marker polypeptide having a GTG startcodon or a TTG startcodon, and wherein the open reading frame sequence that encodes the selectable marker protein has been mutated to replace at least half of its CpG dinucleotides as compared to the native open reading frame sequence that encodes the selectable marker protein. The invention further provides such DNA molecules wherein the open reading frame sequence that encodes a selectable marker polypeptide is part of a multicistronic transcription unit that further comprises an open reading frame sequence encoding a polypeptide of interest. The invention also provides methods for obtaining host cells expressing a polypeptide of interest, said host cells comprising the DNA molecules of the invention. The invention further provides the production of polypeptides of interest, comprising culturing host cells comprising the DNA molecules according to the invention.

1 - 92 . (canceled)  
     
     
         93 . A DNA molecule comprising an open reading frame sequence that encodes a selectable marker polypeptide, wherein said DNA molecule in the coding strand comprises a startcodon for the selectable marker polypeptide chosen from the group consisting of: 
 a) GTG; and    b) TTG;    and wherein the open reading frame sequence that encodes the selectable marker protein has been mutated to replace at least half of its CpG dinucleotides as compared to the native open reading frame sequence that encodes the selectable marker protein.    
     
     
         94 . The DNA molecule of  claim 93 , wherein the the selectable marker polypeptide has a TTG start codon.  
     
     
         95 . The DNA molecule of  claim 93 , wherein the open reading frame sequence that encodes the selectable marker polypeptide has no ATG sequence in the coding strand.  
     
     
         96 . The DNA molecule of  claim 93 , wherein the selectable marker polypeptide provides resistance against zeocin or against neomycin.  
     
     
         97 . The DNA molecule of  claim 96 , comprising an open reading frame sequence that encodes a polypeptide that provides resistance against zeocin, wherein the DNA molecule comprises a sequence chosen from the group consisting of: 
 a) SEQ. ID. NO. 92, with the proviso that at least half of the CpG dinucleotides has been replaced without mutating the amino acid sequence that is encoded, and with the further proviso that the startcodon is either GTG or TTG; and    b) SEQ. ID. NO. 92 wherein nucleotide A at position 280 is replaced by T, and with the proviso that at least half of the CpG dinucleotides has been replaced without mutating the amino acid sequence that is encoded, and with the further proviso that the startcodon is either GTG or TTG.    
     
     
         98 . The DNA molecule of  claim 97 , comprising SEQ. ID. NO. 132.  
     
     
         99 . The DNA molecule of  claim 96 , comprising an open reading frame sequence that encodes a polypeptide that provides resistance against neomycin, wherein the DNA molecule comprises a sequence chosen from the group consisting of: 
 a) SEQ. ID. NO. 128, with the proviso that at least half of the CpG dinucleotides has been replaced without mutating the amino acid sequence that is encoded, and with the further proviso that the startcodon is either GTG or TTG; and    b) SEQ. ID. NO. 118, with the proviso that at least half of the CpG dinucleotides of the coding strand has been replaced without mutating the amino acid sequence that is encoded, and with the further proviso that the startcodon is either GTG or TTG; and    c) SEQ. ID. NO. 128 or SEQ. ID. NO. 118, with the proviso that it contains a mutation to encode either of the following polypeptide variants as compared to the polypeptide encoded by the native sequences: 
 (i) substitution of valine at position 201 into glycine (201V>G), or  
 (ii) subtitution of glutamic acid at position 185 into aspartic acid (185E>D), or  
 (iii) a combination of both mutations (i) and (ii) (185E>D and 201V>G),  
 with the further proviso that at least half of the CpG dinucleotides of the coding strand has been replaced without further mutating the amino acid sequence that is encoded beyond the mutation indicated under (i)-(iii), and with the further proviso that the startcodon is either GTG or TTG.  
   
     
     
         100 . The DNA molecule of  claim 99 , comprising SEQ. ID. NO. 130, with the proviso that nucleotide A at position 555 is replaced by C, and that nucleotide T at position 602 is replaced by G and that nucleotide G at position 603 is replaced by T, and with the further proviso that the startcodon is either GTG or TTG.  
     
     
         101 . The DNA molecule of  claim 93 , wherein 
 the open reading frame sequence that encodes a selectable marker polypeptide is part of a multicistronic transcription unit that further comprises an open reading frame sequence encoding a polypeptide of interest.    
     
     
         102 . The DNA molecule of claim l01, wherein the open reading frame that encodes the selectable marker polypeptide is upstream of the open reading frame encoding the polypeptide of interest, 
 and wherein the open reading frame that encodes the selectable marker polypeptide has no ATG sequence in the coding strand.    
     
     
         103 . The DNA molecule of  claim 101 , wherein the open reading frame encoding the polypeptide of interest is upstream of the open reading frame that encodes the selectable marker polypeptide, 
 and wherein the open reading frame that encodes the selectable marker polypeptide is operably linked to an internal ribosome entry site (IRES).    
     
     
         104 . An expression cassette comprising the DNA molecule of  claim 101 , said expression cassette comprising a promoter upstream of said multicistronic expression unit and a transcription termination sequence downstream of the multicistronic expression unit, wherein said expression cassette is functional in a eukaryotic host cell for initiating transcription of the multicistronic expression unit.  
     
     
         105 . The expression cassette of  claim 104 , further comprising at least one element selected from the group consisting of matrix or scaffold attachment regions (MAR/SAR), and anti-repressor (STAR) sequences.  
     
     
         106 . The expression cassette of  claim 105 , wherein said at least one element is an anti-repressor sequence selected from the group consisting of: 
 a) any one of SEQ. ID. NO. 1 through SEQ. ID. NO. 66 and    b) the complement of a).    
     
     
         107 . The expression cassette of  claim 106 , wherein said expression cassette comprises SEQ. ID. NO. 66 positioned upstream of the promoter that drives transcription of the multicistronic expression unit.  
     
     
         108 . The expression cassette of  claim 106 , wherein said multicistronic expression unit is flanked on both sides by at least one anti-repressor sequence chosen from the group consisting of: 
 a) any one of SEQ. ID. NO. 1 through SEQ. ID. NO. 65 and    b) the complement of a).    
     
     
         109 . A host cell comprising the DNA molecule of  claim 93 .  
     
     
         110 . A host cell comprising the expression cassette of  claim 104 .  
     
     
         111 . A method of generating a host cell able to express a polypeptide of interest, said method comprising the steps of: 
 a) introducing into a plurality of precursor cells a DNA molecule according to  claim 101 , and    b) culturing the plurality of precursor cells under conditions suitable for expression of the selectable marker polypeptide, and    c) selecting at least one host cell expressing the polypeptide of interest.    
     
     
         112 . A method of generating a host cell able to express a polypeptide of interest, said method comprising the steps of: 
 a) introducing into a plurality of precursor cells an expression cassette according to  claim 104 , and    b) culturing the plurality of precursor cells under conditions suitable for expression of the selectable marker polypeptide, and    c) selecting at least one host cell expressing the polypeptide of interest.    
     
     
         113 . A method of expressing a polypeptide of interest, comprising culturing a host cell comprising the expression cassette of  claim 104 , and expressing the polypeptide of interest from the expression cassette.  
     
     
         114 . The method according to  claim 113 , further comprising harvesting the polypeptide of interest.  
     
     
         115 - 140 . (canceled)  
     
     
         141 . A host cell comprising the DNA molecule of  claim 101.