Increased production of a secreted glycoprotein in engineered CHO cells through amplification of a transcription factor.
Expression levels of reporter protein driven by Mouse Mammary Tumor Virus Promoter system were improved by expressing its specific transcription factor (glucocorticoid receptor) from a different expression vector. The vector that expresses glucocorticoid receptor (GR) also contained dihydrofolate reductase (dhfr) gene as a selection marker. In the presentstudy we amplified the glucocorticoid receptor gene (gr)along with the dhfr gene by adapting the cell lines to increasing concentrations of methotrexate, an antifolate analog. Stepwise increases in the volumetric titers of a secreted reporter glycoprotein, Secreted Alkaline Phosphatase (SEAP), were observed in recombinant Chinese hamster ovary (CHO) cellsgrowing in increased concentrations of methotrexate. Western andRT-PCR analysis showed that this increase in volumetric titers is associated with higher levels of GR expressed in CHO cellsgrowing in increased concentration of methotrexate. A stablytransfected cell line growing in 10(-6) M methotrexate wasgrown in suspension culture and induced with 10(-7) Mdexamethasone. The SEAP volumetric titers reached a peak of approximately 23 mug ml(-1) on the 5th day after induction.Inducing these cells with increasing concentrations of dexamethasone resulted in increased specific productivity. These high volumetric productivities were further increased in fed-batch bioreactors.
Mangalampalli
Venkata R M
VR
Department of Chemical Engineering, University of Colorado, Boulder, CO, 80309, USA.
Mowry
Mark C
MC
Lipscomb
Matthew L
ML
James
Rohaizah I
RI
Johnson
Alyssa K
AK
Kompala
Dhinakar S
DS