DOI QR코드

DOI QR Code

Effects of Storage-protein 2 Derived from Silkworm Hemolymph on Reduction of Aggregation and Cell Death in CHO Cells

CHO 세포에서 누에 혈림프 유래 Storage-protein 2의 세포응집 및 세포사멸 억제 효과

  • 임진혁 (인하대학교 공과대학 생물공학과) ;
  • 차현명 (인하대학교 공과대학 생물공학과) ;
  • 김지훈 (차의과학대학교 생명과학대학 바이오공학과) ;
  • 최용수 (차의과학대학교 생명과학대학 바이오공학과) ;
  • 김동일 (인하대학교 공과대학 생물공학과)
  • Received : 2016.01.07
  • Accepted : 2016.03.16
  • Published : 2016.03.31

Abstract

Chinese hamster ovary (CHO) cells have been widely used for production of various recombinant proteins such as cytokines and monoclonal antibodies. The cell aggregation and cell death in CHO cell culture directly affect cell viability, and productivity and quality of products. In this study, we investigated preventing effects of storage-protein 2 (SP2) derived from silkworm hemolymph on cell aggregation and cell death in CHO cell culture producing albuminerythropoietin (Alb-EPO). The viable cell density in the culture supplemented with 2 mg/mL SP2 was 1.71-fold higher than that in control culture. Increased titer of Alb-EPO was also found in the culture with SP2. Morphology of CHO cells in SP2 supplemented cultures did not differ from that of control. In addition, the cell aggregation rate of the SP2 cultures was reduced 20% compared to the control. Finally, we confirmed that the apoptosis was strongly suppressed by addition of SP2 in the cultures. These results clearly demonstrate that SP2 can be served as an effective supplement for enhancing titer of Alb-EPO via reducing cell aggregation and cell death.

Keywords

References

  1. Butler, M. (2005) Animal cell cultures: Recent achievements and perspectives in the production of biopharmaceuticals. Appl. Microbiol. Biotechnol. 68: 283-291. https://doi.org/10.1007/s00253-005-1980-8
  2. Li, F., N. Vijayasankaran, A. Shen, R. Kiss, and A. Amanullah (2010) Cell culture processes for monoclonal antibody production. mAbs. 2: 466-479. https://doi.org/10.4161/mabs.2.5.12720
  3. Li, J., C. L. Wong, N. Vijayasankaran, T. Hudson, and A. Amanullah (2012) Feeding lactate for CHO cell culture processes: Impact on culture metabolism and performance. Biotechnol. Bioeng. 109: 1173-1186. https://doi.org/10.1002/bit.24389
  4. Chen, P. and S. W. Harcum (2005) Effects of amino acid additions on ammonium stressed CHO cells. J. Biotechnol. 117: 277-286. https://doi.org/10.1016/j.jbiotec.2005.02.003
  5. Chuan, K. H., S. F. Lim, L. Martin, C. Y. Yun, S. O. H. Loh, F. Lasne, and Z. Song (2006) Caspase activation, sialidase release and changes in sialylation pattern of recombinant human erythropoietin produced by CHO cells in batch and fed-batch cultures. Cytotechnology. 51: 67-79. https://doi.org/10.1007/s10616-006-9016-5
  6. Kim, Y. J., E. Baek, J. S. Lee, and G. M. Lee (2013) Autophagy and its implication in Chinese hamster ovary cell culture. Biotechnol. Lett. 35: 1753-1763. https://doi.org/10.1007/s10529-013-1276-5
  7. Goswami, J., A. J. Sinskey, H. Steller, G. N. Stephanopoulos, and D. I. C. Wang (1999) Apoptosis in batch cultures of Chinese hamster ovary cells. Biotechnol. Bioeng. 62: 632-640. https://doi.org/10.1002/(SICI)1097-0290(19990320)62:6<632::AID-BIT2>3.0.CO;2-I
  8. Park, J. G., S. S. Choi, and T. H. Park (2007) Enhancement of cell growth and viability of CHO cells in serum-free media by 30Kc6 gene expression. Process Biochem. 42: 8-15. https://doi.org/10.1016/j.procbio.2006.04.010
  9. Figueroa Jr., B., T. M. Sauerwald, G. A. Oyler, J. M. Hardwick, and M. J. Betenbaugh (2003) A comparison of the properties of a Bcl-xL variant to the wild-type anti-apoptosis inhibitor in mammalian cell cultures. Metab. Eng. 5: 230-245. https://doi.org/10.1016/S1096-7176(03)00044-2
  10. Krampe, B. and M. Al-Rubeai (2010) Cell death in mammalian cell culture: Molecular mechanisms and cell line engineering strategies. Cytotechnology. 62: 175-188. https://doi.org/10.1007/s10616-010-9274-0
  11. Halliwell, B. (2014) Cell culture, oxidative stress, and antioxidants: Avoiding pitfalls. Biomed. J. 37: 99-105.
  12. Hossler, P., M. Wang, S. Mcdermott, C. Racicot, K. Chemfe, Y. Zhang, C. Chumsae, and A. Manuilov (2015) Cell culture media supplementation of bioflavonoids for the targeted reduction of acidic species charge variants on recombinant therapeutic proteins. Biotechnol. Prog. 31: 1039-1052. https://doi.org/10.1002/btpr.2095
  13. Chang, K. H., Y. T. Jeong, C. Y. Kwak, O. Choi, and J. H. Kim (2013) Effect of mild-thiol reducing agents and a2,3-sialyltransferase expression on secretion and sialylation of recombinant EPO in CHO cells. J. Microbiol. Biotechnol. 23: 699-706. https://doi.org/10.4014/jmb.1303.03046
  14. Rhee, W. J. and T. H. Park (2000) Silkworm hemolymph inhibits baculovirus-induced insect cell apoptosis. Biochem. Biophys. Res. Commun. 271: 186-190. https://doi.org/10.1006/bbrc.2000.2592
  15. 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. https://doi.org/10.1016/S0006-291X(03)01425-6
  16. Cha, H. M., S. M. Kim, Y. S. Choi, J. S. Park, J. H. Lim, S. G. Hwang, and D. I. Kim (2014) Serum-free media supplement from silkworm Gland for the expansion of mesenchymal stem cells. Tissue. Eng. Regen. Med. 12: 53-59.
  17. Wang, Z., X. Ma, L. Zhao, L. Fan, and W. S. Tan (2012) Expression of anti-apoptotic 30Kc6 gene inhibiting hyperosmotic pressure-induced apoptosis in antibody-producing Chinese hamster ovary cells. Process Biochem. 47: 735-741. https://doi.org/10.1016/j.procbio.2012.02.001
  18. 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. https://doi.org/10.1002/bit.23091
  19. Fujii, T., H. Sakurai, S. Izumi, and S. Tomino (1989) Structure of the gene for the arylphorin-type storage protein SP 2 of Bombyx mori. J. Biol. Chem. 264: 11020-11025.
  20. Yu, W., M. Wang, H. Zhang, Y. Quan, and Y. Zhang (2013) Expression and functional analysis of storage protein 2 in the silkworm, Bombyx mori. Int J Genomics. 2013: 145450
  21. 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. https://doi.org/10.1021/bp0702065
  22. Joung, C. H., J. Y. Shin, J. K. Koo, J. J. Lim, J. S. Wang, S. J. Lee, H. K. Tan, S. L. Kim, and S. M. Lim (2009) Production and characterization of long-acting recombinant human albumin-EPO fusion protein expressed in CHO cell. Protein Expr. Purif. 68: 137-145. https://doi.org/10.1016/j.pep.2009.07.003
  23. Kim, Z. H., J. W. Hwang, J. H. Lee, H. Kim, D. S. Lim, S. Kang, H. S. Lee and Y. S. Choi (2014) Whitening effect of storage protein 2 from silkworm hemolymph. Adv. Biosci. Biotechnol. 5: 758-767. https://doi.org/10.4236/abb.2014.59089
  24. Choi, M. H., H. M. Cha, S. M. Kim, Y. S. Choi and D. I. Kim (2013) Effects of Silkworm Gland Hydrolysate on Albumin-erythropoietin Production in Transgenic Chinese Hamster Ovary Cells. Korean J. Biotechnol. Bioeng. 28: 86-91.
  25. Cavallaro, U. and G. Christofori (2004) Cell adhesion and signalling by cadherins and Ig-CAMs in cancer. Nat. Rev. Cancer. 4: 118-132. https://doi.org/10.1038/nrc1276
  26. Hwang, E. H. (2009) Effects of medium components and culture conditions on cell aggregation in suspension culture of CHO-DG44 cells. M.S. Thesis. Hanyang University, Seoul, Korea.
  27. Circu, M. L. and T. Y. Aw (2010), Reactive oxygen species, cellular redox systems, and apoptosis. Free Radic. Biol. Med. 48: 749-762. https://doi.org/10.1016/j.freeradbiomed.2009.12.022
  28. Mondal, M., K. Trivedy and S. N. Kumar (2007) The silk proteins, sericin and fibroin in silkworm, Bombyx mori Linn., - A review. Caspian J. Env. Sci. 5: 63-76
  29. Park, J. S. (2009) Utilization of storage-protein 2 from silkworm hemolymph for cryopreservation of human mesenchymal stem cells. M.S. Thesis. Inha University, Incheon, Korea.