KSBB Journal

The Korean Society for Biotechnology and Bioengineering (한국생물공학회)
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In situ Recovery of hGM-CSF in Transgenic Rice Cell Suspension Cultures

형질전환 벼 현탁세포 배양에서 hGM-CSF의 in situ Recovery 연구

  • Received : 2015.02.06
  • Accepted : 2015.05.26
  • Published : 2015.06.27
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Abstract

Production of foreign proteins by transgenic plant cell cultures has several advantages such as post-translational modification, low risk of product contamination and low-cost production and purification. However, target proteins are degraded by extracellular proteases existing in the media. A solution to this problem is the use of perfusion culture and ion exchange chromatography for the application of integrated bioprocess using in situ recovery. With this method, production of human granulocyte-macrophage colony-stimulating factor (hGM-CSF) was investigated in this study. First, optimization of cell concentration during the induction phase for the production of hGM-CSF was examined. As cell concentration increased, the level of hGM-CSF was decreased due to the presence of extracellular proteases. Induction using sugarfree media produced 33% more hGM-CSF. The effects of pH on the binding of hGM-CSF to cationic and anionic exchange resins were also investigated. In terms of stability, optimal pH was found to be 5~7. In the case of using buffer exchange when CM-Sepharose was used as a cationic exchange resin, optimal pH for binding was 4.8 and adsorption yield was 77%. When DEAE-Sepharose was used as an anionic exchange resin, it was 5.5 (74%). Without buffer exchange, optimal pH was 4.6 (84%). From these results, an integrated bioprocess using in situ recovery with simultaneous production and separation of foreign protein in transgenic plant cell suspension cultures was found to be feasible.

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References

  1. James, E. A., C. Wang, Z. Wang, R. Reeves, J. H. Shin, N. S. Magnuson, and J. M. Lee (2000) Production and characterization of biologically active human GM-CSF secreted by genetically modified plant cells. Protein Expr. Purif. 19: 131-138. https://doi.org/10.1006/prep.2000.1232
  2. Zhang, X. W., T. Sun, X. Liu, D. X. Gu, and Z. Q. Tang (1999) Production of granulocyte macrophage colony stimulating factor (GM-CSF) by high cell density fermentation of secretory recombination Escherichia coli. Process Biochem. 34: 55-58. https://doi.org/10.1016/S0032-9592(98)00067-3
  3. Shin, Y. J., S. Y. Hong, T. H. Kwon, Y. S. Jang, and M. S. Yang (2003) High level of expression of recombinant human granulocyte- macrophage colony stimulating factor in transgenic rice cell suspension culture. Biotechnol. Bioeng. 82: 778-783. https://doi.org/10.1002/bit.10635
  4. Sharma, A. K. and M. K. Sharma (2009) Plants as bioreactors: Recent developments and emerging opportunities. Biotechnol. Adv. 27: 811-832. https://doi.org/10.1016/j.biotechadv.2009.06.004
  5. Twyman, R. M., E. Stoger, S. Schillberg, P. Christou, and R. Fischer (2003) Molecular farming in plants: Host systems and expression technology. Trends Biotechnol. 21: 570-578. https://doi.org/10.1016/j.tibtech.2003.10.002
  6. Mascia, P. N. and R. B. Flavell (2004) Safe and acceptable strategies for producing foreign molecules in plants. Curr. Opin. Plant. Biol. 7: 189-195. https://doi.org/10.1016/j.pbi.2004.01.014
  7. Grabowski, G. A., M. Golembo, and Y. Shaaltiel (2014) Taliglucerase alfa: An enzyme replacement therapy using plant cell expression technology. Mol. Gen. Metabol. 112: 1-8. https://doi.org/10.1016/j.ymgme.2014.02.011
  8. Magnuson, N. S., P. M. Linzmaier, J.-W. Gao, R. Reeves, G. An, and J. M. Lee (1996) Enhanced recovery of secreted mammalian protein from suspension culture of genetically modified tobacco cells. Protein Expr. Purif. 7: 220-228. https://doi.org/10.1006/prep.1996.0030
  9. Hooker, B. S. and J. M. Lee (1990) Cultivation of plant cells in aqueous two-phase polymer systems. Plant Cell Rep. 8: 546-549. https://doi.org/10.1007/BF00820206
  10. Tjerneld, F., I. Persson, and J. M. Lee (1991) Enzymatic cellulose hydrolysis an attrition bioreactor combined with an aqueous twophase system. Biotechnol. Bioeng. 37: 876-882. https://doi.org/10.1002/bit.260370912
  11. James, E., D. R. Mills, 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
  12. Terashima, M., N. Hashikawa, M. Hattori, and H. Yoshida (2002) Growth characteristics of rice cell genetically modified for recombinant human a1-antitrypsin production. Biochem. Eng. J. 12: 155-160. https://doi.org/10.1016/S1369-703X(02)00064-5
  13. Kwon, J. Y., K. H. Lee, S. H. Cheon, 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. Technol. 50: 298-303. https://doi.org/10.1016/j.enzmictec.2012.02.004
  14. James, E. A. and J. M. Lee (2001) The production of foreign protein from genetically modified plant cells. Adv. Biochem. Eng. Biotechnol. 72: 127-156.
  15. Kusnadi, A. R., Z. L. Nikolov, and J. A. Howard (1997) Production of recombinant protein in transgenic plants: practical considerations. Biotechnol. Bioeng. 56: 473-484. https://doi.org/10.1002/(SICI)1097-0290(19971205)56:5<473::AID-BIT1>3.0.CO;2-F
  16. Magnuson, N. S., P. M. Linzmaier, R. Reeves, G. An, K. Hay-Glass, and J. M. Lee (1998) Secretion of biologically active human interleukin-2 and interleukin-4 from genetically modified tobacco cells in suspension culture. Protein Express. Purif. 13: 45-52. https://doi.org/10.1006/prep.1998.0872
  17. Velez, D., L. Miller, and J. D. Macmillan (1989) Use of tangenitial flow filtration in perfusion propagation of hybridoma cells for production of monoclonal antibodies. Biotechnol. Bioeng. 33: 938-940. https://doi.org/10.1002/bit.260330721
  18. Su, W. W., F. Lei, and N. P. Kao (1995) High density cultivation of Anchusa officinalis in a stirred-tank bioreactor with in situ filtration. Appl. Microbiol. Biotechnol. 44: 293-299. https://doi.org/10.1007/BF00169919
  19. Choi, J. W., G. H. Cho, S. Y. Byun, and D. I. Kim (2001) Integrated bioprocessing for plant cell cultures. Adv. Biochem. Eng. Biotechnol. 72: 63-103.