Functional expression of human pyruvate carboxylase for reduced lactic acid formation of Chinese hamster ovary cells (DG44).
To investigate the effect of human pyruvate carboxylase (hPC) on lactate formation in Chinese hamster ovary (CHO) cell lines, FLAG-tagged hPC was introduced into a dihydrofolate-deficient CHO cell line (DG44). Three clones expressing high levels of hPC, determined by Western blotting using an anti-FLAG monoclonal antibody, and a control cell line were established. Immunocytochemistry revealed that a substantial amount of expressed hPC protein was localized in the mitochondria of the cells. hPC expression did not impair cell proliferation. Rather, it improved cell viability at the end of adherent batch cultures with the serum-containing medium probably because of reduced lactate formation. Compared with control cells, specific lactate production rate of the three clones was decreased by 21-39%, which was because of a decreased specific glucose uptake rate and yield of lactate from glucose. Reduced lactate formation by hPC expression was also observed in suspension fed-batch cultures using a serum-free medium. Taken together, these results demonstrate that through the expression of the hPC enzyme, lactate formation in CHO cell culture can be efficiently reduced.
Kim
Sung Hyun
SH
Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1 Kuseong-Dong, Yuseong-Gu, Daejeon, 305-701, South Korea.
Lee
Gyun Min
GM
D000818:Animals; D019149:Bioreactors; D001709:Biotechnology; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D019344:Lactic Acid / Q000096:biosynthesis*; D008928:Mitochondria / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011766:Pyruvate Carboxylase / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D011994:Recombinant Proteins; D019344:Lactic Acid; D011766:Pyruvate Carboxylase