Selection of host cells expressing protein at high levels using a selectable marker with a non-optimal start codon

摘要

Disclosed is a DNA molecule comprising a polycistronic transcription unit coding for i) a selectable marker polypeptide capable of being selected for 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 said polycistronic transcription unit, and the sequence encoding the selectable maker polypeptide has no ATG sequence in the coding strand following the start codon of the selectable marker polypeptide up to the start codon of the polypeptide of interest, and wherein the translation start sequence in the coding strand for the selectable marker polypeptide is chosen 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 ATG outside the brackets is the start codon; 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.