Selection of host cells expressing protein at high levels

Abstract

The invention provides 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, characterized in that the coding sequence for the polypeptide of interest is downstream from the coding sequence for the selectable marker in said 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. 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 . 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,    wherein the polypeptide of interest has a translation initiation sequence separate from that of the selectable marker polypeptide, and    wherein the coding sequence for the polypeptide of interest is downstream from the coding sequence for the selectable marker in said multicistronic transcription unit, and    wherein the nucleic acid sequence coding for the selectable marker polypeptide in the coding strand comprises a translation start sequence chosen from the group consisting of:    a) an ATG startcodon 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 startcodon is underlined;    b) a GTG startcodon;    c) a TTG startcodon;    d) a CTG startcodon;    e) a ATT startcodon; and    f) a ACG startcodon.    
     
     
         2 . A DNA molecule according to  claim 1 , wherein the translation start sequence in the coding strand for the selectable marker polypeptide comprises a GTG startcodon.  
     
     
         3 . A DNA molecule according to  claim 1 , wherein the translation start sequence in the coding strand for the selectable marker polypeptide comprises a TTG startcodon.  
     
     
         4 . A DNA molecule according to  claim 1 , wherein the sequence encoding the selectable marker polypeptide has no ATG sequence in the coding strand following the startcodon of the selectable marker polypeptide up to the startcodon of the polypeptide of interest.  
     
     
         5 . A DNA molecule according to  claim 4 , wherein any sequence ATG when present in frame and coding for methionine in the sequence coding for the wild type marker polypeptide is mutated to code for an amino acid chosen from the group consisting of valine, threonine, isoleucine, and leucine.  
     
     
         6 . A DNA molecule according to  claim 1 , wherein the selectable marker protein provides resistance against lethal or growth-inhibitory effects of a selection agent.  
     
     
         7 . A DNA molecule according to  claim 6 , wherein said selection agent is an antibiotic.  
     
     
         8 . A DNA molecule according to  claim 6 , wherein said selection agent is chosen from the group consisting of: zeocin, puromycin, blasticidin, hygromycin, neomycin, methotrexate, methionine sulphoximine and kanamycin.  
     
     
         9 . A DNA molecule according to  claim 6 , wherein the selection agent is zeocin.  
     
     
         10 . A DNA molecule according to  claim 6 , wherein the selection agent is blasticidin.  
     
     
         11 . A DNA molecule according to  claim 6 , wherein the selection agent is puromycin.  
     
     
         12 . A DNA molecule according to  claim 6 , wherein the selectable marker protein is encoded by the neomycin resistance gene.  
     
     
         13 . A DNA molecule according to  claim 6 , wherein the selectable marker protein is encoded by the dhfr gene.  
     
     
         14 . A DNA molecule according to  claim 1 , wherein the selectable marker polypeptide further comprises a mutation that reduces the activity of the selectable marker polypeptide compared to its wild-type counterpart.  
     
     
         15 . A DNA molecule according to  claim 14 , wherein the selectable marker polypeptide is a zeocin resistance polypeptide wherein proline at position 9 has been changed into a different amino acid.  
     
     
         16 . A DNA molecule according to  claim 1 , wherein the coding sequence of the polypeptide of interest comprises an optimal translation start sequence, comprising the sequence (A/G)CC ATG G wherein the startcodon is underlined.  
     
     
         17 . An expression cassette comprising a DNA molecule according to  claim 1 , said expression cassette further comprising a promoter upstream of said multicistronic expression unit and being functional in a eukaryotic host cell for initiating transcription of the multicistronic expression unit, and said expression cassette further comprising a transcription termination sequence downstream of the multicistronic expression unit.  
     
     
         18 . An expression cassette according to  claim 17 , further comprising at least one chromatin control element, chosen 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.  
     
     
         19 . An expression cassette according to  claim 18 , wherein said at least one chromatin control element is the chicken lysosyme 5′ MAR.  
     
     
         20 . An expression cassette according to  claim 18 , wherein said chromatin control element is an anti-repressor sequence.  
     
     
         21 . An expression cassette according to  claim 20 , wherein said anti-repressor sequence is chosen from the group consisting of: 
 a) any one of SEQ. ID. NO.1 through SEQ. ID. NO.66;    b) fragments of any one of SEQ. ID. NO. 1 through SEQ. ID. NO. 66, wherein said fragments have anti-repressor activity;    c) sequences that are at least 70% identical in nucleotide sequence to a) or b) wherein said sequences have anti-repressor activity; and    d) the complement to any one of a) to c).    
     
     
         22 . An expression cassette according to  claim 21 , wherein said expression cassette comprises one of: 
 a) SEQ. ID. NO.66,    b) a fragment of a) having anti-repressor activity;    c) a sequence that is at least 70% identical in nucleotide sequence to a) or b) wherein said sequence has anti-repressor activity, wherein a), b) or c) is positioned upstream of the promoter that drives transcription of the multicistronic gene.    
     
     
         23 . An expression cassette according to  claim 17 , wherein said multicistronic gene is flanked on both sides by at least one anti-repressor sequence.  
     
     
         24 . An expression cassette according to  claim 23 , wherein said anti-repressor sequence is chosen from the group consisting of: 
 a) any one of SEQ. ID. NO. 1 through SEQ. ID. NO. 65;    b) fragments of any one of SEQ. ID. NO. 1 through SEQ. ID. NO. 65, wherein said fragments have anti-repressor activity;    c) sequences that are at least 70% identical in nucleotide sequence to a) or b) wherein said sequences have anti-repressor activity; and    d) the complement to any one of a) to c).    
     
     
         25 . An expression cassette according to  claim 23 , comprising: 5′- anti-repressor sequence A—anti-repressor sequence B-promoter—multicistronic gene encoding the functional selectable marker protein and downstream thereof the polypeptide of interest—transcription termination sequence—anti-repressor sequence C-3′, 
 wherein anti-repressor sequences A and C may be the same or different and are chosen from the group consisting of:    a) any one of SEQ. ID. NO. 1 through SEQ. ID. NO. 65;    b) fragments of any one of the sequences of a), said fragments having anti-repressor activity;    c) sequences that are at least 70% identical in nucleotide sequence to a) or b) wherein said sequences have anti-repressor activity,    and wherein anti-repressor sequence B is chosen from the group consisting of:    i) SEQ. ID. NO. 66,    ii) a fragment of i) having anti-repressor activity; and    iii) a sequence that is at least 70% identical in nucleotide sequence to i) or ii) wherein said sequence has anti-repressor activity.    
     
     
         26 . An expression cassette according to  claim 25 , wherein anti-repressor sequences A and C are chosen from: 
 a) SEQ. ID. NO. 7;    b) fragments of one of the sequences of a), said fragments having anti-repressor activity; and    c) sequences that are at least 70% identical in nucleotide sequence to a) or b) wherein said sequences have anti-repressor activity.    
     
     
         27 . An expression cassette according to  claim 17 , wherein the polypeptide of interest is a part of a multimeric protein.  
     
     
         28 . An expression cassette according to  claim 27 , wherein the polypeptide of interest is chosen from the group consisting of an immunoglobulin light chain and an immunoglobulin heavy chain.  
     
     
         29 . An expression cassette according to  claim 17 , further comprising: 
 a) a transcription pause (TRAP) sequence upstream of said promoter and being in a 5′ to 3′ direction; or    b) a TRAP sequence downstream of said transcription termination sequence and being in a 3′ to 5′ orientation; or    c) both a) and b);    wherein a TRAP sequence is a sequence which when placed into a transcription unit, results in a reduced level of transcription in the nucleic acid present on the 3′ side of the TRAP when compared to the level of transcription observed in the nucleic acid on the 5′ side of the TRAP.    
     
     
         30 . An expression cassette according to  claim 30 , wherein said TRAP is SEQ. ID. NO. 142.  
     
     
         31 . A DNA molecule comprising a sequence encoding a selectable marker polypeptide functional in a eukaryotic cell, wherein: 
 i) the sequence encoding the selectable marker polypeptide in the coding strand comprises a translation start sequence chosen from the group consisting of:    a) an ATG startcodon 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 startcodon is underlined;    b) a GTG startcodon;    c) a TTG startcodon;    d) a CTG startcodon;    e) a ATT startcodon; and    f) a ACG startcodon,    and wherein the DNA molecule    ii) in the coding strand of the sequence defining the selectable marker polypeptide downstream of the non-optimal startcodon and up to the stopcodon is devoid of ATG sequences.    
     
     
         32 . A host cell comprising a DNA molecule according to  claim 1 .  
     
     
         33 . A host cell comprising a DNA molecule according to  claim 2 .  
     
     
         34 . A host cell comprising a DNA molecule according to  claim 3 .  
     
     
         35 . A host cell comprising a DNA molecule according to  claim 4 .  
     
     
         36 . A host cell comprising an expression cassette according to  claim 17 .  
     
     
         37 . A host cell comprising an expression cassette according to  claim 18 .  
     
     
         38 . A host cell comprising a DNA molecule according to  claim 31 .  
     
     
         39 . A host cell according to  claim 32 , which is a eukaryotic host cell.  
     
     
         40 . A host cell according to  claim 39 , which is a mammalian host cell.  
     
     
         41 . A host cell according to  claim 40 , being a Chinese Hamster Ovary (CHO) cell.  
     
     
         42 . A method for generating a host cell expressing a polypeptide of interest, the method comprising the steps of: 
 a) introducing into a plurality of precursor cells a DNA molecule according to  claim 1 , and    b) culturing the generated cells under conditions selecting for expression of the selectable marker polypeptide, and    c) selecting at least one host cell producing the polypeptide of interest.    
     
     
         43 . A method for generating a host cell expressing a polypeptide of interest, the method comprising the steps of: 
 a) introducing into a plurality of precursor cells an expression cassette according to  claim 17 , and    b) culturing the generated cells under conditions selecting for expression of the selectable marker polypeptide, and    c) selecting at least one host cell producing the polypeptide of interest.    
     
     
         44 . A method for producing a polypeptide of interest, comprising culturing a host cell, said host cell comprising an expression cassette according to  claim 17 , and expressing the polypeptide of interest from the expression cassette.  
     
     
         45 . A method for producing a polypeptide of interest, comprising culturing a host cell, said host cell comprising an expression cassette according to  claim 18 , and expressing the polypeptide of interest from the expression cassette.  
     
     
         46 . A method according to  claim 44 , further comprising isolating the polypeptide of interest.  
     
     
         47 . A method according to  claim 45 , further comprising isolating the polypeptide of interest.  
     
     
         48 . An RNA molecule having the sequence of the transcription product of a DNA molecule according to  claim 1 .  
     
     
         49 . A selectable marker polypeptide encoded by a DNA molecule according to  claim 31 , wherein the selectable marker polypeptide provides resistance against lethal or growth-inhibitory effects of a selection agent chosen from the group consisting of: zeocin, puromycin, blasticidin, hygromycin, neomycin, methotrexate, methionine sulphoximine, and kanamycin, with the proviso that the selectable marker polypeptide is not the blasticidine resistance protein having methionine as first amino acid (SEQ. ID. NO. 111).  
     
     
         50 . A DNA molecule comprising a sequence encoding a polypeptide conferring resistance to zeocin in eukaryotic cells, wherein the sequence in the coding strand comprises a translation start sequence chosen from the group consisting of: 
 a) an ATG startcodon 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 startcodon is underlined;    b) a GTG startcodon;    c) a TTG startcodon;    d) a CTG startcodon;    e) a ATT startcodon; and    f) a ACG startcodon.    
     
     
         51 . A DNA molecule according to  claim 50 , wherein the sequence in the coding strand comprises a GTG startcodon.  
     
     
         52 . A DNA molecule according to  claim 50 , wherein the sequence in the coding strand comprises a TTG startcodon.  
     
     
         53 . A DNA molecule comprising a sequence encoding a polypeptide conferring resistance to blasticidin in eukaryotic cells, wherein the sequence in the coding strand comprises a translation start sequence chosen from the group consisting of: 
 a) an ATG startcodon 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 startcodon is underlined;    b) a GTG startcodon;    c) a TTG startcodon;    d) a CTG startcodon;    e) a ATT startcodon; and    f) a ACG startcodon.    
     
     
         54 . A DNA molecule according to  claim 53 , wherein the sequence in the coding strand comprises a GTG startcodon.  
     
     
         55 . A DNA molecule according to  claim 53 , wherein the sequence in the coding strand comprises a TTG startcodon.  
     
     
         56 . An expression cassette according to  claim 17 , wherein said promoter is a CMV promoter.  
     
     
         57 . An expression cassette according to  claim 17 , wherein said promoter is an SV40 promoter.  
     
     
         58 . A DNA molecule comprising two expression cassettes according to  claim 17 , wherein the first expression cassette encodes an antibody heavy chain as polypeptide of interest, and wherein the second expression cassette encodes an antibody light chain as polypeptide of interest, and wherein the first and second expression cassette encode different selectable marker polypeptides.  
     
     
         59 . A DNA molecule according to  claim 58 , further comprising a functional promoter operably linked to a sequence encoding dhfr.  
     
     
         60 . A method for producing an antibody, the method comprising culturing a eukaryotic cell and harvesting the antibody from the cells in the culture or from the culture supernatant or from both the cells and the culture supernatant, 
 wherein the cell comprises a first 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 the heavy chain of the antibody, and    wherein the second multicistronic transcription unit encodes a second functional selectable marker polypeptide and the light chain of the antibody, and    wherein the heavy and light chain of the antibody have translation initiation sequences separate from that of the first and second selectable marker polypeptides respectively, and    wherein the coding sequences for the heavy and light chains of the antibody are downstream of the coding sequences of the first and second functional selectable markers in the first and second multicistronic transcription units respectively, and    wherein the sequences encoding the first and second selectable marker polypeptides have no ATG sequence in the coding strands following the startcodons of the selectable marker polypeptides up to the startcodons of the heavy and light chain respectively, and    wherein the sequences coding for the first and second selectable marker polypeptide in the coding strand comprise a translation start sequence each independently chosen from the group consisting of:    a) an ATG startcodon in a non-optimal context for translation initiation, comprising the sequence (C/T)(AIT/G)(A/T/G) ATG (A/T/C) wherein the startcodon is underlined;    b) a GTG startcodon;    c) a TTG startcodon;    d) a CTG startcodon;    e) a ATT startcodon; and    f) a ACG startcodon.