Optimised mammalian expression through the coupling of codon adaptation with gene amplification: maximum yields with minimum effort.
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.
Biopharm Process Research, Biopharm R&D, GlaxoSmithKline, Stevenage SG1 2NY, UK. nina.a.kotsopoulou@gsk.com
Ekaterini
E
Kotsopoulou
Hella
H
Bosteels
Yuen-Ting
YT
Chim
Pamela
P
Pegman
George
G
Stephen
Susannah I
SI
Thornhill
James D
JD
Faulkner
Mark
M
Uden
D000818:Animals; D016466:CHO Cells; D003062:Codon; D006224:Cricetinae; D003412:Cricetulus; D005784:Gene Amplification; D018628:Gene Dosage; D008727:Methotrexate; D016366:Open Reading Frames; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase
D003062:Codon; D011994:Recombinant Proteins; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate