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Query: EC:6.4.1.1 (pyruvate carboxylase)
 1,516 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Recently, a recombinant yeast pyruvate carboxylase expressed in the cytoplasm of BHK-21 cells was shown to reconstitute the missing link between glycolysis and TCA, thus increasing the flux of glucose into the TCA and resulting in a higher intracellular ATP content. Now, these metabolically engineered cells have been additionally transfected with a plasmid bearing the gene for human erythropoietin. EPO yield and substrate-specific productivity of the recombinant BHK-21 cells have been compared to control cells without the PYC2-gene but transfected with the plasmid coding for the expression of the selection genes and EPO. PYC2-expressing clones showed a 2-fold higher glucose-specific productivity and a 2-fold higher product concentration in a continuously perfused bioreactor. Moreover, the PYC2 expression enabled the cells to become more resistant to low glucose concentrations in the culture medium. They could produce at nearly maximum productivity under glucose-limiting conditions of 0.05-1 gl(-1) that guaranteed a reduced accumulation of lactate in fed-batch production systems. Due to the fact that PYC2-expressing cells are characterized by reduced glucose consumption, a prolonged production phase in bioreactors can be maintained. Based on the demand not to fall short of 80% cell viability for the production, EPO could be produced for 2 days (30%) longer compared to the control due to a more economic exploitation of glucose, and the prolonged viability period of the cells using a batch cultivation driven by glutamine limitation.
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PMID:Expression of recombinant cytoplasmic yeast pyruvate carboxylase for the improvement of the production of human erythropoietin by recombinant BHK-21 cells. 1175 90

Recently, we demonstrated that a recombinant yeast pyruvate carboxylase expressed in the cytoplasm of BHK-21 cells was shown to partially reconstitute the missing link between glycolysis and TCA, increasing the flux of glucose into the TCA and achieving higher yields of recombinant erythropoietin. In the present study, a CHO cell line producing recombinant human granulocyte macrophage colony stimulating factor was used to evaluate the impact of PYC2 expression and reduced culture temperature. Temperature reduction from 37 to 33 degrees C revealed a reduced growth rate, a prolonged stationary phase and a 2.1-fold increase of the cell specific rhGM-CSF production rate for CHO-K1-hGM-CSF cells. The PYC2-expressing cell clones showed a decreased cell growth and a lower maximum cell concentration compared to the control expressing rhGM-CSF but no PYC2. However, only 65% lactate were produced in PYC2-expressing cells and the product yield was 200% higher compared to the control. The results obtained for CHO cells compared to BHK cells reported previously, indicated that the PYC2 expression dominantly reduced the lactate formation and increased the yield of the recombinant protein to be produced. Finally, the growth and productivity of PYC2-expressing CHO-K1-hGM-CSF cells under both temperature conditions were investigated. The average cell specific rhGM-CSF production increased by 3.2-fold under reduced temperature conditions. The results revealed that the expression of PYC2 and a reduced culture temperature have an additive effect on the cell specific productivity of CHO-K1-hGM-CSF cells.
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PMID:Impact of temperature reduction and expression of yeast pyruvate carboxylase on hGM-CSF-producing CHO cells. 1506 26