Selection of host cells expressing protein at high levels

摘要

The invention provides a DNA molecule comprising a multicistronic transcription unit coding for i) a polypeptide of interest, and for ii) a selectable marker polypeptide functional in a eukaryotic host cell, 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 upstream from the coding sequence for the selectable marker polypeptide in the multicistronic transcription unit, and wherein an internal ribosome entry site is present downstream from the coding sequence for the polypeptide of interest and upstream from the coding sequence for the selectable marker polypeptide, and wherein the nucleic acid sequence coding for the selectable marker polypeptide in the coding strand comprises a GTG or a TTG start codon. Also provided are methods for obtaining host cells expressing a polypeptide of interest, such host cells comprising DNA molecules of the invention. Further provided is the production of polypeptides of interest, comprising culturing host cells comprising the DNA molecules of the invention.

主权项

1. A DNA molecule comprising a multicistronic transcription unit, wherein the multicistronic transcription unit comprises, from 5′ to 3′:
a first anti-repressor (STAR) element that is STAR7 (SEQ ID NO:7), an anti-repressor (STAR) element that is STAR67 (SEQ ID NO:66), a polynucleotide encoding a first selectable marker polypeptide, wherein the polynucleotide encoding the first selectable marker polypeptide comprises a translation initiation sequence separate from that of a polynucleotide encoding a polypeptide of interest, does not comprise an ATG codon in the coding strand between the start codon of the first selectable marker polypeptide and the start codon of the polypeptide of interest, and comprises a GTG start codon or a TTG start codon, the polynucleotide encoding the polypeptide of interest, wherein the polynucleotide encoding the polypeptide of interest has a translation initiation sequence separate from that of a polynucleotide encoding a second selectable marker polypeptide, an internal ribosome entry site (IRES), the polynucleotide encoding a second selectable marker polypeptide, wherein the polynucleotide encoding the second selectable marker polypeptide is under translational control of the IRES and comprises a GTG start codon, a TTG start codon, a CTG start codon, an ATT start codon or an ACG start codon, a transcription terminator, and a second anti-repressor (STAR) element that is STAR7 (SEQ ID NO:7).