[Metabolism of recombinant CHO-GS cell reducing of toxic effect of ammonia].
The toxic effect of ammonia on rCHO-GS cell decreased obviously due to the transfection of GS system in serum-free culture. The maximum cell density, 15.6 x 10(5) cells/mL was obtained in the culture with 1.42 mmol/L ammonia. The growth of rCHO-GS cell was inhibited with an increased ammonia concentration. However, a cell density of 8.9 x 10(5) cells/mL was obtained when the concentration of ammonia was 12.65mmol/L. The intracellar metabolic pathways were affected due to the decrease of the toxic effect of ammonia on rCHO-GS cell. With the increase of initial ammonia concentration from 0.36mmol/L to 12.65mmol/L, the yield coefficients of cell to glucose and lactate to glucose decreased. The activities of hexokinase (HK), pyruvate kinase (PK), and lactate dehydrogenase (LDH) increased by 43%, 140% and 25%, respectively, indicating that the utilization of glucose increased and the glycolysis pathway was more prone to efficient energy metabolism pathway. An increased activity of glutamate-pyruvate aminotransferase (GPT) showed that the conversation from glutamate to alpha-ketoglutarate was shifted to glutamate-pyruvate transamination pathway. The deamination pathway was inhibited due to a decreased activity of glutamate dehydrogenase. In addition, the number of cells in G0/G1 phase increased and the specific production rate of recombinant protein increased by 2.1-fold with the increase of initial ammonia concentration from 0.36mmol/L to 12.65mmol/L.
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China.
Fang
F
Zhang
Xiao-Ping
XP
Yi
Xiang-Ming
XM
Sun
Yuan-Xing
YX
Zhang
D000641:Ammonia / Q000378:metabolism / Q000633:toxicity*; D000818:Animals; D016466:CHO Cells; D018929:Cell Culture Techniques / Q000379:methods*; D006224:Cricetinae; D003412:Cricetulus; D016895:Culture Media, Serum-Free; D005818:Genetic Engineering / Q000379:methods; D005974:Glutamate-Ammonia Ligase / Q000235:genetics* / Q000378:metabolism; D005973:Glutamine / Q000378:metabolism
D016895:Culture Media, Serum-Free; D005973:Glutamine; D000641:Ammonia; D005974:Glutamate-Ammonia Ligase