DOI QR코드

DOI QR Code

In situ Recovery of hCTLA4Ig from Suspension Cell Cultures of Oryza sativa

형질전환 벼 현탁세포 배양에서 hCTLA4Ig의 in situ 회수

  • Choi, Hong-Yeol (Department of Biological Engineering, Inha University) ;
  • Cheon, Su-Hwan (Department of Life Science, Gachon University) ;
  • Kwon, Jun-Young (Department of Biological Engineering, Inha University) ;
  • Yun, Boreum (Department of Biological Engineering, Inha University) ;
  • Hong, Seok-Mi (Department of Biological Engineering, Inha University) ;
  • Kim, Sun-Dal (Department of Biological Engineering, Inha University) ;
  • Kim, Dong-Il (Department of Biological Engineering, Inha University)
  • Received : 2016.08.31
  • Accepted : 2016.12.23
  • Published : 2016.12.31

Abstract

In this research, recombinant human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) was produced by transgenic rice cells. RAmy3D promoter was used for overcome the limitation of low expression level in transgenic plant cells, and the secretion of target protein was accomplished by signal peptide. However, the RAmy3D promoter system which can be induced only by sugar starvation causes the decrease of cell viability. As a result, cell death promotes the release of protease which degrades the target proteins. The protein stability and productivity can be significantly influenced by proteolysis activity. Therefore, development of new strategies are necessary for the in situ recovery of target proteins from cell culture media. In this study, in situ recovery was performed by various strategies. Direct addition of Protein A resin with nylon bag leads to cell death by increased shear stress and decrease in production of hCTLA4Ig by protease. Medium exchange through modified flask could recover hCTLA4Ig with high cell viability and low protease activity, on the other hand, the productivity was lower than that of control. When in situ recovery was conducted at day 7 after induction in air-lift bioreactor, 1.94-fold of hCTLA4Ig could be recovered compared to control culture without in situ recovery. Consequently, in situ recovery of hCTLA4Ig from transgenic rice cell culture could enhance productivity significantly and prevent degradation of target proteins effectively.

Keywords

References

  1. 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. and D.-I. Kim (2007) Production and characterization of human CTLA4Ig expressed in transgenic rice cell suspension cultures. Protein Expres. Purif. 51: 293-302. https://doi.org/10.1016/j.pep.2006.08.019
  2. W. R. Strohl (2015) Fusion proteins for half-life extension of biologics as a strategy to make biobetters. Biodrugs 29: 215-239. https://doi.org/10.1007/s40259-015-0133-6
  3. Toyofuku, K., Umemura, T.-A. and J. Yamaguchi (1998) Promoter elements required for sugar-repression of the RAmy3D gene for ${\alpha}$-amylase in rice. FEBS Lett. 428: 275-280. https://doi.org/10.1016/S0014-5793(98)00518-3
  4. Takatsuka, C., Inoue, Y., Matsuoka, K. and Y. Moriyasu (2004) 3-Methyladenine inhibits autophagy in tobacco culture cells under sucrose starvation conditions. Plant Cell Physiol. 45: 265-274. https://doi.org/10.1093/pcp/pch031
  5. Kwon, J.-Y., Lee, K.-H., Cheon, S.-H. and D.-I. Kim (2012) Application of anoxia with glucose addition for the enhanced production of hCTLA4Ig in transgenic rice suspension cell cultures. Enzyme Microb. Tech. 50: 298-303. https://doi.org/10.1016/j.enzmictec.2012.02.004
  6. Woodley, J. M., Bisschops, M., Straathof, A. J. J. and M. Ottens (2008) Future directions for in-situ product removal (ISPR). J. Chem. Technol. Biot. 83: 121-123. https://doi.org/10.1002/jctb.1790
  7. James, E., Mills, D. R. and J. M. Lee (2002) Increased production and recovery of secreted foreign proteins from plant cell cultures using an affinity chromatography bioreactor. Biochem. Eng. J. 12: 205-213. https://doi.org/10.1016/S1369-703X(02)00073-6
  8. J. F. Buyel (2015) Process development strategies in plant molecular farming. Curr. Pharm. Biotechnol. 16: 966-982. https://doi.org/10.2174/138920101611150902115413
  9. He, X., Haselhorst, T., von Itzstein, M., Kolarich, D., Packer, N. H., Gloster, T. M., Vocadlo, D. J., Clarke, L. A., Qian, Y., and A. R. Kermode (2012) Production of ${\alpha}$-L-iduronidase in maize for the potential treatment of a human lysosomal storage disease. Nat. Commun. 3: 1062. https://doi.org/10.1038/ncomms2070
  10. Jung, J.-W., Kim, N.-S., Jang, S.-H., Shin, Y.-J. and M.-S. Yang (2016) Production and characterization of recombinant human acid ${\alpha}$-glucosidase in transgenic rice cell suspension culture. J. Biotechnol. 226: 44-53. https://doi.org/10.1016/j.jbiotec.2016.03.031
  11. Kuo, Y.-C., Tan, C.-C., Ku, J.-T., Hsu, W.-C., Su, S.-C., Lu, C.-A. and L.-F. Huang (2013) Improving pharmaceutical protein production in Oryza sativa. Int. J. Mol. Sci. 14: 8719-8739. https://doi.org/10.3390/ijms14058719
  12. Stark, D. and U. von Stockar (2003) In situ product removal (ISPR) in whole cell biotechnology during the last twenty years. Adv. Biochem. Eng. Biot. 80: 149-175.
  13. Komaraiah, P., Ramakrishna, S. V., Reddanna, P. and P. B. Kavi Kishor (2003) Enhanced production of plumbagin in immobilized cells of Plumbago rosea by elicitation and in situ adsorption. J. Biotechnol. 101: 181-187. https://doi.org/10.1016/S0168-1656(02)00338-3
  14. Roja, G., Bhangale, A. S., Juvekar, A. R., Eapen, S. and S. F. D'Souza (2005) Enhanced production of the polysaccharide arabinogalactan using immobilized cultures of Tinospora cordifolia by elicitation and in situ adsorption. Biotechnol. Progr. 21: 1688-1691. https://doi.org/10.1021/bp050188w
  15. Chakrabarti, S. K., Matsumura, N. and R. S. Ranu (2000) Purification and characterization of an extracellular alkaline serine protease from Aspergillus terreus (IJIRA 6.2). Curr. Microbiol. 40: 239-244. https://doi.org/10.1007/s002849910048
  16. Kwon, J.-Y., Cheon, S.-H., Nam, H.-J., Choi, H.-Y. and D.-I. Kim (2013) Process characterization of hCTLA4Ig production in transgenic rice cell cultures using a 3-L bioreactor. Appl. Biochem. Biotech. 171: 1276-1288. https://doi.org/10.1007/s12010-013-0192-4