# US 8,771,984 B2 · Selection of host cells expressing protein at high levels > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 8,771,984 B2 - Document ID: US8771984B2 - Status: Granted - Kind: B2 - Application number: 12/807,133 - Filed: 2010-08-26 - Published: 2011-01-20 - Granted: 2014-07-08 - Earliest claimed: 2004-11-08 - Assignees: Chromagenics B.V. - Applicants: Arie P. Otte; Henricus J. M. van Blokland; Theodorus H. J. Kwaks; Richard G. A. B. Sewalt - Inventors: Arie P. Otte; Henricus J. M. van Blokland; Theodorus H. J. Kwaks; Richard G. A. B. Sewalt - Web page: https://patentgrep.com/patent/US8771984B2 ## CPC - C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies - C07K 14/00: Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof - C07K 16/30: from tumour cells - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) ## 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 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. The invention also provides methods for obtaining host cells expressing a polypeptide of interest, the 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. ## Claims ### Claim 1 A method for utilizing a multicistronic transcription unit to produce a peptide of interest from a host cell, the method comprising: introducing into a plurality of host cells of a eukaryotic cell line a DNA molecule comprising, in 5′-3′ direction: a promoter, a bicistronic transcription unit comprising: a first upstream polynucleotide with the translation start codon selected from the group consisting of GTG and TTG, wherein the first upstream polynucleotide encodes a selectable marker polypeptide with no methionine amino acids in its sequence that provides resistance against the lethal or growth-inhibitory effects of a selection marker, and a second downstream polynucleotide encoding the peptide of interest, wherein the second downstream polynucleotide has the translation initiation sequence ATG, wherein the bicistronic transcription unit has no ATG sequence between the start codon of the first upstream polynucleotide and the start codon of the second downstream polynucleotide, and a transcription termination sequence, wherein the DNA molecule further comprises at least two anti-repressor (STAR) sequences flanking the bicistronic transcription unit, wherein the at least two anti-repressor (STAR) sequences flanking the bicistronic transcription unit are a STAR7 sequence (SEQ ID NO:7) and a STAR67 sequence (SEQ ID NO:66) located upstream of the promoter, and a STAR7 sequence (SEQ ID NO:7) located downstream of the transcription termination sequence; culturing the plurality of host cells to select for expression of the selectable marker polypeptide; selecting at least one host cell producing the peptide of interest; and producing the peptide of interest from the host cell. ### Claim 2 A recombinant expression vector comprising in a 5′-3′ direction: a promoter, a bicistronic transcription unit comprising: a first upstream polynucleotide with the translation start codon selected from the group consisting of GTG and TTG, wherein the first upstream polynucleotide encodes a selectable marker polypeptide with no methionine amino acids in its sequence that provides resistance against the lethal or growth-inhibitory effects of a selection marker, and a second downstream polynucleotide encoding the peptide of interest, wherein the second downstream polynucleotide has the translation initiation sequence ATG, wherein the bicistronic transcription unit has no ATG sequence between the start codon of the first upstream polynucleotide and the start codon of the second downstream polynucleotide, and a transcription termination sequence, wherein the recombinant expression vector further comprises at least two anti-repressor (STAR) sequences, wherein the at least two anti-repressor (STAR) sequences flanking the bicistronic transcription unit are a STAR 7 sequence (SEQ ID NO:7) and a STAR67 sequence (SEQ ID NO:66) located upstream of the promoter, and a STAR 7 sequence (SEQ ID NO:7) located downstream of the transcription termination sequence.