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• W2056133764 startingPage "1558" @default.
• W2056133764 abstract "The CHEF1 expression plasmid utilizes regulatory domains of the Chinese Hamster Elongation Factor 1 (CHEF1) housekeeping gene to drive stable recombinant protein expression in Chinese Hamster Ovary (CHO) cells. Rapid development of stable CHEF1 cell lines is possible, in part, because high titer pools are produced in the absence of methotrexate-induced DHFR amplification. Inhibiting DHFR activity with methotrexate increases the selection pressure on transfected cells and can result in a compensatory event to ensure cell survival, such as dhfr gene amplification or integration into a transcriptionally active genomic site. Methotrexate amplification often results in improved cell line productivity but it is a time-consuming process. Herein, we describe a novel mechanism to increase selection stringency via codon deoptimization of the mouse dhfr selectable marker utilizing hamster codon preference that results in improved expression of a linked recombinant protein compared to wild-type DHFR selection. We show that deoptimizing the translatability of the dhfr gene reduces the expression level of the DHFR protein in CHO transfection pools and derived clones. Lower DHFR expression increases the transfection selection stringency, shown as lower transfection efficiency in pools, and correlates with increased recombinant protein expression in both pools and clones. These results demonstrate a new mechanism for increasing selection stringency and improving recombinant protein production during cell line development without time-consuming gene amplification steps." @default.
• W2056133764 created "2016-06-24" @default.
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• W2056133764 date "2010-10-14" @default.
• W2056133764 modified "2023-10-01" @default.
• W2056133764 title "Improved recombinant protein yield using a codon deoptimized DHFR selectable marker in a CHEF1 expression plasmid" @default.
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• W2056133764 doi "https://doi.org/10.1002/btpr.491" @default.
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