Optimised mammalian expression through the coupling of codon adaptation with gene amplification: Maximum yields with minimum effort
| Autor: | George Stephen, Pamela Pegman, James D. Faulkner, Yuen-Ting Chim, Mark Uden, Hella Bosteels, Ekaterini Kotsopoulou, Susannah I. Thornhill |
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| Rok vydání: | 2010 |
| Předmět: |
Mammalian expression
Gene Dosage Bioengineering CHO Cells Computational biology Applied Microbiology and Biotechnology Gene dosage law.invention Open Reading Frames Cricetulus law Cricetinae Dihydrofolate reductase Gene duplication Animals Codon Gene Genetics biology Chinese hamster ovary cell Gene Amplification General Medicine Recombinant Proteins Tetrahydrofolate Dehydrogenase Open reading frame Methotrexate biology.protein Recombinant DNA Biotechnology |
| Zdroj: | Journal of Biotechnology. 146:186-193 |
| ISSN: | 0168-1656 |
| DOI: | 10.1016/j.jbiotec.2010.02.004 |
| Popis: | Gene amplification methodologies are frequently employed for the generation of large quantities of recombinant proteins in mammalian cells. Although they usually guarantee very high yields, they are very time consuming. In addition, due to the large genomic re-arrangements that frequently occur with amplification, the resulting high-producing clones can be unstable. We herein describe significant improvements to the dihydrofolate reductase (DHFR)/methotrexate (MTX) based gene amplification methodology typically employed to improve yields of recombinant proteins produced in genetically engineered CHO host cells. We demonstrate substantial synergy when such gene amplification is combined with extremely high codon optimisation strategies. As a result, expression saturation can be achieved rapidly, in as low as 5 nM MTX, with minimal effort and without compromise in final yields achieved. |
| Databáze: | OpenAIRE |
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