Selection of host cells expressing protein at high levels

Details Selection of host cells expressing protein at high levels Selection of host cells expressing protein at high levels

C - Chemistry, Metallurgy

12

N

C12N 15/67 (2006.01) C12N 5/10 (2006.01) C12N 15/09 (2006.01) C12N 15/11 (2006.01) C12N 15/79 (2006.01) C12N 15/85 (2006.01)

Patent

CA 2581422

Title: Selection of host cells expressing protein at high levels Abstract The invention provides a DNA molecule comprising a 5 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 10 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 15 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 20 the invention. The invention further provides the production of polypeptides of interest, comprising culturing host cells comprising the DNA molecules according to the invention.

L'invention concerne une molécule d'ADN, qui comprend une unité de transcription multicistronique codant pour: i) un polypeptide marqueur sélectable agissant dans une cellule hôte eucaryote; et ii) un polypeptide d'intérêt dont la séquence d'initiation de traduction est distincte de celle du polypeptide marqueur sélectable. La molécule d'ADN se caractérise en ce que la séquence de codage pour le polypeptide d'intérêt se trouve en aval de la séquence de codage pour le polypeptide marqueur sélectable dans ladite unité de transcription multicistronique, et que la séquence d'acides nucléiques codant pour le polypeptide marqueur sélectable comprend une mutation qui diminue l'efficacité de traduction du marqueur sélectable dans la cellule hôte eucaryote. L'invention concerne également des méthodes de production de cellules hôtes exprimant un polypeptide d'intérêt, lesquelles cellules hôtes comprennent les molécules d'ADN de l'invention. L'invention concerne en outre la production de polypeptides d'intérêt, qui consiste à cultiver des cellules hôtes comprenant les molécules d'ADN de l'invention.

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