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Improved recombinant protein yield using a codon deoptimized DHFR selectable marker in a CHEF1 expression plasmid.

Amy D Westwood1, Daniel A Rowe, Howard R G Clarke

  • 1CMC ICOS Biologics, 22021 20th Avenue SE, Bothell, WA 98021, USA.

Biotechnology Progress
|October 16, 2010
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Chinese Hamster Elongation Factor 1 (CHEF1) gene regulatory elements enhance recombinant protein production in Chinese Hamster Ovary (CHO) cells. Codon deoptimization of the DHFR selectable marker improves expression and selection stringency without gene amplification.

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Area of Science:

  • Biotechnology
  • Molecular Biology
  • Cell Line Development

Background:

  • Chinese Hamster Ovary (CHO) cells are widely used for recombinant protein production.
  • Stable cell line development often relies on selection markers like DHFR, typically amplified using methotrexate.
  • Methotrexate-induced DHFR amplification is effective but time-consuming.

Purpose of the Study:

  • To develop a novel method for increasing selection stringency in CHO cell line development.
  • To improve recombinant protein expression without lengthy gene amplification steps.
  • To investigate the effect of DHFR codon deoptimization on selection and protein production.

Main Methods:

  • Utilized Chinese Hamster Elongation Factor 1 (CHEF1) regulatory elements for stable expression.
  • Engineered a mouse DHFR selectable marker with codon deoptimization based on hamster codon preference.
  • Assessed transfection efficiency, DHFR protein levels, and recombinant protein expression in CHO pools and clones.

Main Results:

  • Codon deoptimization of the DHFR gene reduced DHFR protein expression in CHO cells.
  • Reduced DHFR expression increased selection stringency, indicated by lower transfection efficiency in pools.
  • This approach correlated with significantly increased recombinant protein expression in both pools and clones.
  • Achieved improved cell line productivity without methotrexate-induced DHFR amplification.

Conclusions:

  • Codon deoptimization of selectable markers is a novel strategy to enhance selection stringency.
  • This method accelerates cell line development and improves recombinant protein yield in CHO systems.
  • Offers an alternative to traditional, time-intensive gene amplification techniques for cell line engineering.