Abstract
Silkworm contains several distinct proteins that have anti-apoptotic activities in mammalian and insect cells. Although the characteristics of 30K proteins have been well investigated, those of SPs (Storage Proteins) have not been examined. In this article, an anti-apoptotic effect of SP1 was investigated in HeLa cell apoptosis. SP1, a type of insect hemocyanin protein, is a female-specific protein in silkworm and consists of high portion of methionine. Transient expression of SP1 in HeLa cells slightly increased cell viability after the treatment with staurosporine, which induced apoptosis via intrinsic mitochondrial pathway. To investigate the anti-apoptotic effect of SP1, the stable cell line expressing SP1 were screened and constructed. The anti-apoptotic effect of the cell line was assessed using trypan blue exclusion assay, microscopic analysis of nuclear fragmentation, and flow cytometry. SP1 expressing cell line showed strong antiapoptotic activity against the treatment of staurosporine but not TNFa, meaning that SP1 is a protein inhibitor of apoptosis caused by intrinsic apoptosis pathway. As the SP1 is the novel anti-apoptotic protein, the regulation of apoptosis by SP1 will offer a new strategy that can be utilized in biopharmaceutical industry when the focus is on minimization of cell death and maximization of productivity.
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References
Meier, P., A. Finch, and G. Evan (2000) Apoptosis in development. Nature 407: 796–801.
Thibaud, T. R. and M. Stéphen (2011) Bax: Addressed to kill. Biochimie 93: 1379–1391.
Michael, O. H. (2000) The biochemistry of apoptosis. Nature 407: 770–776.
Favaloro, B., N. Allocati, V. Graziano, C. D. Ilio, and V. D. Laurenzi (2012) Role of apoptosis in disease. Aging 4: 330–349.
Wong, D. C., K. T. Wong, Y. Y. Lee, P. N. Morin, C. K. Heng, and M. G. Yap (2006) Transcriptional profiling of apoptotic pathways in batch and fed-batch CHO cell cultures. Biotechnol. Bioeng. 94: 373–382.
Rao, R. V., H. M. Ellerby, and D. E. Bredesen (2004) Coupling endoplasmic reticulum stress to the cell death program. Cell Death Diff. 11: 372–380.
Antonsson, B., S. Montessuit, B. Sanchez, and J. C. Martinou (2001) Bax is present as a high molecular weight oligomer/complex in the mitochondrial membrane of apoptotic cells. J. Biol. Chem. 276: 11615–11623.
Eskes, R., S. Desagher, B. Antonsson, and J. C. Martinou (2000) Bid induces the oligomerization and insertion of Bax into the outer mitochondrial membrane. Mol. Cell Biol. 20: 929–935.
Brustovetsky, T., T. Li, Y. Yang, B. Antonsson, N. Brustovetsky, and J. T. Zhang (2010) Bax insertion, oligomerization, and outer membrane permeabilization in brain mitochondria: role of permeability transition and SH-redox regulation. Biochim. Biophys. Acta 1797: 1795–1806.
deZengotita, V. M., L. R. Abston, A. E. Schmelzer, S. Shaw, and W. M. Miller (2002) Selected amino acids protect hybridoma and CHO cells from elevated carbon dioxide and osmolality. Biotechnol. Bioeng. 78: 741–752.
Oh, H. K., M. K. So, J. Yang, H. C. Yoon, J. S. Ahn, J. M. Lee, J. T. Kim, J. U. Yoo, and T. H. Byun (2005) Effect of N-acetylcystein on butyrate-treated chinese hamster ovary cells to improve the production of recombinant human interferon-ß-1a. Biotechnol. Prog. 21: 1154–1164.
Rhee, W. J., E. H. Lee, and T. H. Park (2009) Expression of Bombyx mori 30Kc19 protein in Escherichia coli and its anti-apoptotic effect in Sf9 cell. Biotechnol. Bioproc. Eng. 14: 645–650.
Tintó, A., C. Gabernet, J. Vives, E. Prats, J. J. Cairó, L. Cornudella, and F. Gòdia (2002) The protection of hybridoma cells from apoptosis by caspase inhibition allows culture recovery when exposed to non-inducing conditions. J. Biotechnol. 95: 205–214.
Zanghi, J. A., W. A. Renner, J. E. Bailey, and M. Fussenegger (2000) The growth factor inhibitor suramin reduces apoptosis and cell aggregation in protein-free CHO cell batch cultures. Biotechnol. Prog. 16: 319–325.
Wang, Z., J. H. Park, H. H. Park, W. Tan, and T. H. Park (2011) Enhancement of recombinant human EPO production and sialylation in Chinese hamster ovary cells through Bombyx mori 30Kc19 gene expression. Biotechnol. Bioeng. 108: 1634–1642.
Wang, Z., X. Ma, L. Fan, W. J. Rhee, T. H. Park, L. Zhao, and W. S. Tan (2012) Understanding the mechanistic roles of 30Kc6 gene in apoptosis and specific productivity in antibody-producing Chinese hamster ovary cells. Appl. Microbiol. Biotechnol. 94: 1243–1253.
Wu, S. C. (2009) RNA interference technology to improve recombinant protein production in Chinese hamster ovary cells. Biotechnol. Adv. 27: 417–422.
Wong, D. C., K. T. Wong, P. M. Nissom, C. K. Heng, and M. G. Yap (2006) Targeting early apoptotic genes in batch and fedbatch CHO cell cultures. Biotechnol. Bioeng. 95: 350–361.
Yu, W., H. Ying, F. Tong, C. Zhang, Y. Quan, and Y. Zhang (2013) Protective effect of the silkworm protein 30Kc6 on human vascular endothelial cells damaged by oxidized low density lipoprotein (Ox-LDL). PLoS One 8: e68746.
Rhee, W. J., E. J. Kim, and T. H. Park (1999) Kinetic effect of silkworm hemolymph on the delayed host cell death in an insect cell-baculovirus system. Biotechnol. Prog. 15: 1028–1032.
Rhee, W. J., E. J. Kim, and T. H. Park (2002) Silkworm hemolymph as a potent inhibitor of apoptosis in Sf9 cells. Biochem. Biophys. Res. Commun. 295: 779–783.
Kim, E. J., W. J. Rhee, and T. H. Park (2001) Isolation and Characterization of an Apoptosis-Inhibiting Component from the Hemolymph of Bombyx mori. Biochem. Biophys. Res. Commun. 285: 224–228.
Rhee, W. J., E. H. Lee, J. H. Park, J. E. Lee, and T. H. Park (2007) Inhibition of HeLa cell apoptosis by storage-protein 2. Biotechnol. Prog. 23: 1441–1446.
Rhee, W. J. and T. H. Park (2000) Silkworm hemolymph inhibits baculovirus-induced insect cell apoptosis. Biochem. Biophys. Res. Commun. 271: 186–190.
Kim, E. J. and T. H. Park (2003) Anti-apoptosis engineering. Biotechnol. Bioproc. Eng. 8: 76–82.
Ha, S. H., T. H. Park, and S. E. Kim (1996) Silkworm hemolymph as a substitute for fetal bovine serum in insect cell culture. Biotechnol. Tech. 10: 401–406.
Ha, S. H. and T. H. Park (1997) Efficient production of recombinant protein in Spodoptera frugiperda AcNPV system utilizing silkworm hemolymph. Biotechnol. Lett. 19: 1087–1091.
Kim, E. J., H. J. Park, and T. H. Park (2003) Inhibition of apoptosis by recombinant 30K protein originating from silkworm hemolymph. Biochem. Biophys. Res. Commun. 308: 523–528.
Park, H. J., E. J. Kim, T. Y. Koo, and T. H. Park (2003) Purification of recombinant 30K protein produced in Escherichia coli and its anti-apoptotic effect in mammalian and insect cell systems. Enz. Microb. Technol. 33: 466–471.
Choi, S. S., W. J. Rhee, E. J. Kim, and T. H. Park (2006) Enhancement of recombinant protein production in Chinese hamster ovary cells through anti-apoptosis engineering using 30Kc6 gene. Biotechnol. Bioeng. 95: 459–467.
Joosten, C. E., T. H. Park, and M. L. Shuler (2003) Effect of silkworm hemolymph on N-linked glycosylation in two Trichoplusia ni insect cell lines. Biotechnol. Bioeng. 83: 695–705.
Park, J. H., Z. Wang, H. J. Jeong, H. H. Park, B. G. Kim, W. S. Tan, S. S. Choi, and T. H. Park (2012) Enhancement of recombinant human EPO production and glycosylation in serum-free suspension culture of CHO cells through expression and supplementation of 30Kc19. Appl. Microbiol. Biotechnol. 96: 671–683.
Choi, S. S., W. J. Rhee, and T. H. Park (2005) Beneficial effect of silkworm hemolymph on CHO cell system: Inhibition of apoptosis and increase of EPO production. Biotechnol. Bioeng. 91: 793–800.
Beintema, J. J., W. T. Stam, B. Hazes, and M. P. Smidt (1994) Evolution of arthropod hemocyanins and insect storage proteins (Hexamerins). Mol. Biol. Evol. 11: 493–503.
Elmore, S. (2007) Apoptosis: A review of programmed cell death. Toxicol. Pathol. 35: 495–516.
Rhee, W. J., J. H. Park, J. S. Hahn, and T. H. Park (2013) Antiapoptotic mechanism of silkworm hemolymph in HeLa cell apoptosis. Proc. Biochem. 48: 1375–1380.
Park, H. H., Y. Sohn, J. W. Yeo, J. H. Park, H. J. Lee, J. Ryu, W. J. Rhee, and T. H. Park (2014) Dimerization of 30Kc19 protein in the presence of amphiphilic moiety and importance of Cys-57 during cell penetration. Biotechol. J. 9: 1582–1593.
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Lee, J.H., Park, T.H. & Rhee, W.J. Inhibition of apoptosis in HeLa cell by silkworm storage protein 1, SP1. Biotechnol Bioproc E 20, 807–813 (2015). https://doi.org/10.1007/s12257-015-0152-7
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DOI: https://doi.org/10.1007/s12257-015-0152-7