Selection of host cells expressing protein at high levels

Abstract

Provided is a DNA molecule comprising a multicistronic transcription unit coding for i) a selectable marker polypeptide functional in a eukaryotic host cell, and for ii) a polypeptide of interest, the polypeptide of interest having a translation initiation sequence separate from that of the selectable marker polypeptide, wherein the coding sequence for the polypeptide of interest is downstream from the coding sequence for the selectable marker in the multicistronic transcription unit, and the nucleic acid sequence coding for the selectable marker polypeptide comprises a mutation that decreases the translation efficiency of the selectable marker in a eukaryotic host cell. Also provided are methods for obtaining host cells expressing a polypeptide of interest, the host cells comprising the described DNA molecules. Further provided is the production of polypeptides of interest, comprising culturing host cells comprising the DNA molecules hereof.

1 .- 60 . (canceled) 
     
     
         61 . A method for producing a peptide of interest, the method comprising:
 culturing a eukaryotic host cell comprising a DNA molecule comprising, in 5′-3′ direction, a promoter-a multicistronic transcription unit-a transcription termination sequence, wherein the multicistronic transcription unit encodes:   i) a selectable marker polypeptide functional in a eukaryotic host cell; and   ii) a peptide of interest;   wherein the peptide of interest has a translation initiation sequence separate from the selectable marker polypeptide's translation initiation sequence separate; and   wherein the coding sequence for the peptide of interest is downstream from the coding sequence for the selectable marker in the multicistronic transcription unit; and   wherein the sequence encoding the selectable marker polypeptide has no ATG sequence in a coding strand following the start codon of the selectable marker polypeptide up to the start codon of the peptide of interest; and   wherein the polynucleotide encoding the selectable marker polypeptide in the coding strand comprises a translation start sequence selected from the group consisting of a) an ATG start codon in a non-optimal context for translation initiation, comprising the sequence (C/T)(A/T/G)(A/T/G)ATG(A/T/C) wherein the start codon is italicized, b) a GTG start codon, c) a TTG start codon, d) a CTG start codon, e) an ATT start codon, and f) an ACG start codon; and   
       expressing the peptide of interest. 
     
     
         62 . The method according to  claim 61 , further comprising isolating the peptide of interest. 
     
     
         63 . The method according to  claim 61 , wherein the translation start sequence in the polynucleotide encoding the selectable marker polypeptide comprises a GTG start codon. 
     
     
         64 . The method according to  claim 61 , wherein the translation start sequence in the polynucleotide encoding the selectable marker polypeptide comprises a TTG start codon. 
     
     
         65 . The method according to  claim 61 , wherein any ATG, when present in frame and encoding methionine in the sequence encoding wild-type marker polypeptide, is mutated to encode an amino acid selected from the group consisting of valine, threonine, isoleucine, and leucine. 
     
     
         66 . The method according to  claim 61 , wherein the selectable marker protein provides resistance against lethal or growth-inhibitory effects of a selection agent. 
     
     
         67 . The method according to  claim 66 , wherein the selection agent is an antibiotic. 
     
     
         68 . The method according to  claim 66 , wherein the selection agent is selected from the group consisting of a bleocin, puromycin, blasticidin, hygromycin, neomycin, methotrexate, methionine sulphoximine and kanamycin. 
     
     
         69 . The method according to  claim 66 , wherein the selection agent is a bleocin. 
     
     
         70 . The method according to  claim 61 , wherein the DNA molecule further comprises an element selected from the group consisting of a matrix or scaffold attachment region (MAR/SAR), an insulator sequence, a universal chromatin opening element (UCOE), and an anti-repressor (STAR) sequence. 
     
     
         71 . The method according to  claim 70 , further comprising isolating the peptide of interest. 
     
     
         72 . The method according to  claim 70 , wherein the element is an anti-repressor sequence. 
     
     
         73 . The method according to  claim 70 , wherein the element is a matrix attachment region. 
     
     
         74 . A method for producing an antibody, the method comprising:
 culturing a eukaryotic cell comprising a first multicistronic transcription unit and a second multicistronic transcription unit each under control of an independent promoter, wherein the first multicistronic transcription unit encodes a first functional selectable marker polypeptide and heavy chain of the antibody, and wherein the second multicistronic transcription unit encodes a second functional selectable marker polypeptide and light chain of the antibody, and   harvesting antibody from the eukaryotic cell in a culture or from the culture supernatant or from both the eukaryotic cell and the culture supernatant;   wherein the coding sequences for the heavy and light chains have translation initiation sequences separate from the translation initiation sequences for the first and second selectable marker polypeptides respectively,   wherein the coding sequences for the heavy and light chains of the antibody are downstream from the coding sequences of the first and second functional selectable markers in the first and second multicistronic transcription units respectively;   wherein the sequences encoding the first and second selectable marker polypeptides have no ATG sequence in the coding strands following the start codons of the selectable marker polypeptides up to the start codons of the heavy and light chain, respectively; and   wherein the sequences encoding the first and second selectable marker polypeptide in the coding strand comprise a translation start sequence each independently selected from the group consisting of a) an ATG start codon in a non-optimal context for translation initiation, comprising the sequence (C/T)(A/T/G)(A/T/G)ATG(A/T/C) wherein the start codon is italicized, b) a GTG start codon, c) a TTG start codon, d) a CTG start codon, e) an ATT start codon, and f) an ACG start codon.   
     
     
         75 . The method according to  claim 74 , wherein any ATG, when present in frame and encoding methionine in the sequence encoding wild-type marker polypeptide, is mutated to encode an amino acid residue selected from the group consisting of valine, threonine, isoleucine, and leucine. 
     
     
         76 . A method for generating a host cell expressing a peptide of interest, the method comprising:
 introducing into a plurality of eukaryotic precursor cells a DNA molecule comprising a multicistronic transcription unit encoding:
 i) a selectable marker polypeptide functional in a eukaryotic host cell; and 
 ii) a peptide of interest; 
 wherein the peptide of interest has a translation initiation sequence separate from that of the selectable marker polypeptide; 
 wherein the coding sequence for the peptide of interest is downstream from the coding sequence for the selectable marker in the multicistronic transcription unit; 
 wherein the sequence encoding the selectable marker polypeptide has no ATG sequence in a coding strand following the start codon of the selectable marker polypeptide up to the start codon of the peptide of interest; and 
 wherein the polynucleotide encoding the selectable marker polypeptide in the coding strand comprises a translation start sequence selected from the group consisting of a) an ATG start codon in a non-optimal context for translation initiation, comprising the sequence (C/T)(A/T/G)(A/T/G)ATG(A/T/C) wherein the start codon is italicized, b) a GTG start codon, c) a TTG start codon, d) a CTG start codon, e) an ATT start codon; and f) an ACG start codon; 
   culturing the generated cells under conditions selecting for expression of the selectable marker polypeptide; and   selecting at least one host cell producing the peptide of interest.   
     
     
         77 . The method according to  claim 76 , wherein any ATG, when present in frame and encoding methionine in the sequence encoding wild-type marker polypeptide, is mutated to encode an amino acid selected from the group consisting of valine, threonine, isoleucine, and leucine. 
     
     
         78 . The method according to  claim 76 , wherein the DNA molecule further comprises a promoter upstream of the multicistronic transcription unit and a transcription termination sequence downstream of the multicistronic transcription unit.