Selection of host cells expressing protein at high levels

Abstract

Described is a DNA molecule comprising an open reading frame sequence that encodes a selectable marker polypeptide, wherein the DNA molecule in the coding strand comprises a translation start sequence for the selectable marker polypeptide having a GTG start codon or a TTG start codon, and wherein the ORF sequence that encodes the selectable marker protein has been mutated to replace at least half of its CpG dinucleotides as compared to the native ORF sequence that encodes the selectable marker protein. Further provided are such DNA molecules wherein the ORF 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. Also described are methods for obtaining host cells expressing a polypeptide of interest, wherein the host cells comprise the DNA molecules described herein. Further provided is the production of polypeptides of interest, comprising culturing host cells comprising the DNA molecules described herein.

1 .- 92 . (canceled) 
     
     
         93 . A DNA molecule comprising an open reading frame encoding a selectable marker polypeptide, wherein the DNA molecule in the coding strand comprises a translation start sequence for the selectable marker polypeptide selected from the group consisting of:
 a) a GTG start codon; and   b) a TTG start codon;   and wherein the open reading frame encoding the selectable marker protein has been mutated to replace at least half of its CpG dinucleotides as in comparison to the native open reading frame encoding the selectable marker protein.   
     
     
         94 . The DNA molecule of  claim 93 , wherein the translation start sequence for the selectable marker polypeptide comprises a TTG start codon. 
     
     
         95 . The DNA molecule of  claim 94 , wherein the open reading frame encoding the selectable marker polypeptide has no ATG sequence in the coding strand. 
     
     
         96 . The DNA molecule of  claim 95 , wherein the selectable marker polypeptide provides resistance against ZEOCIN® antibiotic or against neomycin. 
     
     
         97 . The DNA molecule of  claim 96 , comprising an open reading frame encoding a polypeptide that provides resistance against ZEOCIN® antibiotic, wherein the DNA molecule comprises a sequence selected 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 start codon 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 start codon is either GTG or TTG. 
 
     
     
         98 . (canceled) 
     
     
         99 . The DNA molecule of  claim 96 , comprising an open reading frame encoding a polypeptide that provides resistance against neomycin, wherein the DNA molecule comprises a sequence selected 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 start codon 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 start codon 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 in comparison to the polypeptide encoded by the native sequences:
 (i) substitution of valine at position 201 into glycine (201V>G), or 
 (ii) substitution 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 start codon 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 start codon is either GTG or TTG. 
     
     
         101 . The DNA molecule of  claim 93 , wherein
 the open reading frame encoding a selectable marker polypeptide is part of a multicistronic transcription unit that further comprises an open reading frame polynucleotide encoding a polypeptide of interest.   
     
     
         102 . The DNA molecule of  claim 101 , wherein the open reading frame encoding the selectable marker polypeptide is upstream of the open reading frame encoding the polypeptide of interest,
 and wherein the open reading frame encoding 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 encoding the selectable marker polypeptide, and
 wherein the open reading frame encoding the selectable marker polypeptide is operably linked to an internal ribosome entry site (IRES).   
     
     
         104 . An expression cassette comprising the DNA molecule of  claim 103 , the expression cassette comprising a promoter upstream of the multicistronic expression unit and a transcription termination sequence downstream of the multicistronic expression unit, wherein the 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 chromatin control 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 the at least one chromatin control element is an anti-repressor molecule selected from the group consisting of any one of SEQ ID NO:1 through SEQ ID NO:66 and the complement of any one of SEQ ID NO:1 through SEQ ID NO:66. 
     
     
         107 . The expression cassette of  claim 106 , wherein the 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 107 , wherein the multicistronic expression unit is flanked on both sides by at least one anti-repressor molecule selected from the group consisting of any one of SEQ ID NO:1 through SEQ ID NO:65 and the complement of any one of SEQ ID NO:1 through SEQ ID NO:65. 
     
     
         109 . A host cell comprising the DNA molecule of  claim 103 . 
     
     
         110 . A host cell comprising the expression cassette of  claim 108 . 
     
     
         111 . A method of generating a host cell able to express a polypeptide of interest, the method comprising the steps of:
 a) introducing into a plurality of precursor cells a DNA molecule according to  claim 103 , 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, the 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)