- Email: [email protected]
Effect of BCL-2 overexpression on HEK293 EBNA cells in transient gene expression system
Abstracts / Journal of Biotechnology 136S (2008) S130–S139
cells against stress and to prevent apoptosis. The aim of this study was to decrease the loss of productivity. Proline has an established role in defending against various stresses with its properties as a compatible solute benefiting a broad range of organisms. The protective role of proline was investigated by examining whether proline supplementation could suppress apoptosis in transgenic rice cell cultures. Various concentrations (1, 2, 3, 4, 5 mM) of proline were added to the media to determine the optimal level. The addition of proline increased cell viability and suppressed apoptosis. Consequently, proline supplementation resulted in an increase of hCTLA4Ig productivity compared with control cultures. References Beers, E.P., Woffenden, B.J., Zhao, C., 2000. Plant proteolytic enzymes: possible roles during programmed cell death. Plant Mol. Biol. 44, 399–415. Chen, C., Dickman, M.B., 2005. Proline suppresses apoptosis in the fungal pathogen of Colletotrichum trifolii. Proc. Natl. Acad. Sci. U.S.A. 102, 3459–3464. Doran, P.M., 2006. Foreign protein degradation and instability in plants and plant tissue cultures. Trends Biotechnol. 21, 426–432. Krishnan, N., Dickman, M.B., Becker, D.F., 2008. Proline modulates the intracellular redox environment and protects mammalian cells against oxidative stress. Free Radic. Biol. Med. 44, 671–681. Lee, S.J., Park, C.I., Park, M.Y., Jung, H.S., Ryu, W.S., Lim, S.M., Tan, H.K., Kwon, T.H., Yang, M.S., Kim, D.I., 2007. Production and characterization of human CTLA4Ig expressed in transgenic rice cell suspension cultures. Protein Expr. Purif. 51, 293–302.
doi:10.1016/j.jbiotec.2008.07.295 II3-YP-018 Effect of BCL-2 overexpression on HEK293 EBNA cells in transient gene expression system So Jeong Lee ∗ , Gyun Min Lee Department of Biological Sciences, KAIST, Daejeon, Republic of Korea E-mail address: [email protected] (S.J. Lee). Most current methods for the production of recombinant proteins are dependent on a stable expression system using mammalian cells in which the gene of interest is stably integrated into the chromosome. However, this technique requires a considerable amount of time and labor. Therefore, a transient gene expression system has been developed for the rapid and cost-effective production of recombinant proteins. HEK293 EBNA cells are widely used in the TGE system as they are highly susceptible to transfection and
S139
easy to grow Sun et al., 2006. Yet there is an obvious disadvantage in this technology such as lower productivity when compared to stable cell lines Wulhfard et al., 2008. Sodium butyrate(NaBu) has been reported to enhance protein production. Regardless of the positive effect of NaBu on the expression of recombinant proteins, it has some drawbacks in that it can inhibit cell growth, which induces apoptotic cell death Kim and Lee, 2000-2001. Consequently, the positive effect of NaBu on protein expression is compromised by its cytotoxicity on cell growth. Previous studies suggest that cytotoxic effect of NaBu can be overcome by overexpression of survival genes such as bcl-2 and bcl-XL Kim and Lee, 2000-2001. Hence, we constructed stable HEK293E-Bcl-2 cell lines which overexpress the human bcl-2 gene to overcome the cytotoxic effect of NaBu added to a suspension culture medium in an attempt to achieve high-level production of humanized antibodies. There are many anti-apoptotic cell lines using CHO cells and HeLa cells, but there has not been any report on HEK293E cells expressing anti-apoptotic genes. Therefore, we concluded that a Bcl-2 overexpressing HEK293E cell line could be a good host in the TGE system in regard of its anti-apoptotic behavior and susceptibility to transient transfection. Here, we investigated the influence of NaBu on cell growth and transient protein production in serum-free suspension culture. In addition, we also evaluated apoptotic cell death induced by NaBu with cell cycle analysis, DNA fragmentation and Western blotting of PARP. Our results show that Bcl-2 cell lines showed higher cell viability and considerably decreased apoptotic cell death compared to the control cell lines which did not overexpress the bcl-2 gene. Bcl-2 overexpression leads to higher protein productivity due to extended culture longevity and enhanced transcription efficiency. References Kim, N.S., Lee, G.M., 2000-2001. Overexpression of bcl-2 inhibits sodium butyrateinduced apoptosis in Chinese hamster ovary cells resulting in enhanced humanized antibody production. Biotechnol. Bioeng. 71 (3), 184–193. Sun, X., Goh, P.E., Wong, T.K., Mori, T., Yap, M.G., 2006. Enhancement of transient gene expression by fed-batch culture of HEK 293 EBNA1 cells in suspension. Biotechnol. Lett. 28, 843–848. Wulhfard, S., Tissot, S., Bouchet, S., Cevey, J., Jesus, M.D., Hacker, D.L., Wurm, F.M., 2008. Mild hypothermia improves transient gene expression yields several fold in Chinese hamster ovary cells. Biotechnol. Prog. 24 (March–April (2)), 458–465.
doi:10.1016/j.jbiotec.2008.07.296
cells against stress and to prevent apoptosis. The aim of this study was to decrease the loss of productivity. Proline has an established role in defending against various stresses with its properties as a compatible solute benefiting a broad range of organisms. The protective role of proline was investigated by examining whether proline supplementation could suppress apoptosis in transgenic rice cell cultures. Various concentrations (1, 2, 3, 4, 5 mM) of proline were added to the media to determine the optimal level. The addition of proline increased cell viability and suppressed apoptosis. Consequently, proline supplementation resulted in an increase of hCTLA4Ig productivity compared with control cultures. References Beers, E.P., Woffenden, B.J., Zhao, C., 2000. Plant proteolytic enzymes: possible roles during programmed cell death. Plant Mol. Biol. 44, 399–415. Chen, C., Dickman, M.B., 2005. Proline suppresses apoptosis in the fungal pathogen of Colletotrichum trifolii. Proc. Natl. Acad. Sci. U.S.A. 102, 3459–3464. Doran, P.M., 2006. Foreign protein degradation and instability in plants and plant tissue cultures. Trends Biotechnol. 21, 426–432. Krishnan, N., Dickman, M.B., Becker, D.F., 2008. Proline modulates the intracellular redox environment and protects mammalian cells against oxidative stress. Free Radic. Biol. Med. 44, 671–681. Lee, S.J., Park, C.I., Park, M.Y., Jung, H.S., Ryu, W.S., Lim, S.M., Tan, H.K., Kwon, T.H., Yang, M.S., Kim, D.I., 2007. Production and characterization of human CTLA4Ig expressed in transgenic rice cell suspension cultures. Protein Expr. Purif. 51, 293–302.
doi:10.1016/j.jbiotec.2008.07.295 II3-YP-018 Effect of BCL-2 overexpression on HEK293 EBNA cells in transient gene expression system So Jeong Lee ∗ , Gyun Min Lee Department of Biological Sciences, KAIST, Daejeon, Republic of Korea E-mail address: [email protected] (S.J. Lee). Most current methods for the production of recombinant proteins are dependent on a stable expression system using mammalian cells in which the gene of interest is stably integrated into the chromosome. However, this technique requires a considerable amount of time and labor. Therefore, a transient gene expression system has been developed for the rapid and cost-effective production of recombinant proteins. HEK293 EBNA cells are widely used in the TGE system as they are highly susceptible to transfection and
S139
easy to grow Sun et al., 2006. Yet there is an obvious disadvantage in this technology such as lower productivity when compared to stable cell lines Wulhfard et al., 2008. Sodium butyrate(NaBu) has been reported to enhance protein production. Regardless of the positive effect of NaBu on the expression of recombinant proteins, it has some drawbacks in that it can inhibit cell growth, which induces apoptotic cell death Kim and Lee, 2000-2001. Consequently, the positive effect of NaBu on protein expression is compromised by its cytotoxicity on cell growth. Previous studies suggest that cytotoxic effect of NaBu can be overcome by overexpression of survival genes such as bcl-2 and bcl-XL Kim and Lee, 2000-2001. Hence, we constructed stable HEK293E-Bcl-2 cell lines which overexpress the human bcl-2 gene to overcome the cytotoxic effect of NaBu added to a suspension culture medium in an attempt to achieve high-level production of humanized antibodies. There are many anti-apoptotic cell lines using CHO cells and HeLa cells, but there has not been any report on HEK293E cells expressing anti-apoptotic genes. Therefore, we concluded that a Bcl-2 overexpressing HEK293E cell line could be a good host in the TGE system in regard of its anti-apoptotic behavior and susceptibility to transient transfection. Here, we investigated the influence of NaBu on cell growth and transient protein production in serum-free suspension culture. In addition, we also evaluated apoptotic cell death induced by NaBu with cell cycle analysis, DNA fragmentation and Western blotting of PARP. Our results show that Bcl-2 cell lines showed higher cell viability and considerably decreased apoptotic cell death compared to the control cell lines which did not overexpress the bcl-2 gene. Bcl-2 overexpression leads to higher protein productivity due to extended culture longevity and enhanced transcription efficiency. References Kim, N.S., Lee, G.M., 2000-2001. Overexpression of bcl-2 inhibits sodium butyrateinduced apoptosis in Chinese hamster ovary cells resulting in enhanced humanized antibody production. Biotechnol. Bioeng. 71 (3), 184–193. Sun, X., Goh, P.E., Wong, T.K., Mori, T., Yap, M.G., 2006. Enhancement of transient gene expression by fed-batch culture of HEK 293 EBNA1 cells in suspension. Biotechnol. Lett. 28, 843–848. Wulhfard, S., Tissot, S., Bouchet, S., Cevey, J., Jesus, M.D., Hacker, D.L., Wurm, F.M., 2008. Mild hypothermia improves transient gene expression yields several fold in Chinese hamster ovary cells. Biotechnol. Prog. 24 (March–April (2)), 458–465.
doi:10.1016/j.jbiotec.2008.07.296