KSBB Journal

The Korean Society for Biotechnology and Bioengineering (한국생물공학회)
권호 표지

Effect of Osmotic Pressure on hCTLA-lg Production in Transgenic Rice Cell Suspension Cultures

형질전환된 벼세포 배양에 있어서 삼투압 조절에 따른 hCTLA4-lg 생산성 변화

  • Choi Sung-Hun (Department of Biological Engineering, Inha University) ;
  • Lee Song-Jae (Boryung Central Research institute, Boryung Pharmaceutical Co. Ltd.) ;
  • Hong Seok-Mi (Department of Biological Engineering, Inha University) ;
  • Cho Ji-Suk (Department of Biological Engineering, Inha University) ;
  • Kim Dong-Il (Department of Biological Engineering, Inha University)
  • 최성훈 (인하대학교 공과대학 생명화학공학부) ;
  • 이송재 ((주)보령제약 중앙연구소) ;
  • 홍석미 (인하대학교 공과대학 생명화학공학부) ;
  • 조지숙 (인하대학교 공과대학 생명화학공학부) ;
  • 김동일 (인하대학교 공과대학 생명화학공학부)
  • Published : 2005.08.01
Download PDF
⟨ Previous Next ⟩

Abstract

An immunosuppressive agent, human cytotoxic T lymphocyte antigen 4 (hCTLA4), is used for the prevention of graft rejection and treatment of autoimmune diseases. hCTLA4-Ig, a CTLA4-immunoglobulin fusion protein, was produced and secreted from transgenic rice cell suspension cultures using rice a-amylase (RAmy3D) expression system. In this system, hCTLA4-Ig expression was regulated metabolically by sugar starvation. For the purpose of improving hCTLA4-Ig production, the effects of osmotic pressure was investigated in suspension cultures of transgenic rice cells. The highest production level was achieved at 40 mM sorbitol $(140\;mOsm{\cdot}kg^{-1}\;H_2O)$. Using the medium with 8 mM glucose, the level of hCTLA4-Ig in the medium reached 45.3 mg/L. By adjusting the osmotic pressure of induction medium, it was found that the hCTLA4-Ig production could be increased up to 2.1-fold compared with that in batch culture.

식물세포의 느린 생장과 낮은 생산량의 이유로 지금까지 는 주로 미생물이나 동물세포에서 유전자 재조합 단백질을 생산하여 왔다. 그러나 저렴한 배지 가격, 동물 유래 바이러스 감염 위험성으로부터의 안정성, glycosylation 등의 post-translational modification이 가능하다는 장점들로 인하여 최근 들어 식물세포배양은 생물학적 활성을 가진 고부가가치의 단백질을 생산하는데 많이 이용되고 있다. 본 연구에서는 생장배지에 첨가했던 sucrose의 소비와 induction 배지로의 교환에서 오는 배지내의 삼투압을 조절하여 hCTLA4-Ig의 생산성을 높이고자 하였다. 다양한 삼투압 조절제 첨가 실험을 통해 sorbitol을 선별하고, 40 mM의 sorbitol 첨가에서 상대적으로 높은 생존도와 induction 후 7일째 대조구보다 1.7배 높은 생산성을 확인하였다. 또한, 저농도의 glucose 첨가를 통한 생산성 증대에 있어서는 8 mM glucose에서 induction 이후에도 높은 세포농도를 유지하면서 최대 37.3 mg/L까지 hCTLA4-Ig 생산량을 증가시켰다. 5-L bioreactor에서 회분식 배양과 induction시의 hCTLA4-Ig 생산량을 비교한 결과 induction시 배양 18일째 최고 45.3 mg/L까지 높일 수 있었으며, 회분식 배양에 비해 2.1배 증가됨을 확인하였다.

Keywords

References

  1. Kusnadi, A. R., Z. L. Nikolov, and J. A. Howard (1997), Production of recombinant proteins 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
  2. Terashima M., Y. Ejiri, N. Hashikawa, and H. Yoshida (1999), Effect of osmotic pressure on human $a_1$-antitrysin production by plant cell culture, Biochem. Eng. J. 4, 31-36 https://doi.org/10.1016/S1369-703X(99)00036-4
  3. Buchner J. and R. Rudoph (1991), Renaturaion, purification and characterization of recombinant Fab-fragments produced in Escherichia coli, BioI. Technol. 9, 157-162 https://doi.org/10.1038/nbt0291-157
  4. Doran, P. M. (2000), Foreign protein production in plant tissue cultures, Curr. Opin. Biotechnol. 11, 199-204 https://doi.org/10.1016/S0958-1669(00)00086-0
  5. Gomord, V. and L. Faye (2004), Posttranslational modification of therapeutic proteins in plants, Curr. Opin. Plant BioI. 7, 171-181 https://doi.org/10.1016/j.pbi.2004.01.015
  6. Matsumoto, S., K. Ikura, M. Veda, and R. Sasaki (1995), Characterization of a human glycoprotein (erythropoietin) produced in cultured tobacco cells, Plant Mol. BioI. 27, 1163-1172 https://doi.org/10.1007/BF00020889
  7. Miele, L. (1997), Plants as bioreactors for biopharmaceutical: regulatory considerations, Trends Biotechnol. 15, 45-50 https://doi.org/10.1016/S0167-7799(97)84202-3
  8. James, E. A. and J. M. Lee (2001), The production of foreign protein from genetically modified plant cells, In: Advances in Biochentical Engineeringl Biotechnology- Plant Cells, (Eds. J. J. Zhong), 72: 127-156 Springer, Berlin, Germany
  9. Sharp, J. M. and P. M. Doran (2001), Characterization of monoclonal antibody fragments produced by plant cells, Biotechnol. Bioeng. 73, 338-346 https://doi.org/10.1002/bit.1067
  10. Taka-aki V., P. Pierdomenico, F. Yuzoo, and Y. Junji (1998), Sugar sensing and a-amylase gene repression in rice embryos, Planta 204, 420-428 https://doi.org/10.1007/s004250050275
  11. Terashima M., Y. Murai, M. Kawamura, S. Nakanishi, T. Stoltz, L. Chen, W. Drohan, R. L. Rodriguez, and S. Kotoh (1999), Production of functional human ai-antitrypsin by plant cell culture, Appl. Microbiol. Biotechnol. 52, 516-523 https://doi.org/10.1007/s002530051554
  12. Shin, Y. J., S. Y. Hong, T. H. Kwon, Y. S. J., and M. S. Yang (2003), High Level of expression of recombinant human granulocyte-marcrophage colony stimulating factor in transgenic rice cell suspension culture, Biotechnol. Bioeng. 82, 778-783 https://doi.org/10.1002/bit.10635
  13. Lui, V. C. H., P. K. H. Tam, M. Y. K. Leung, J. Y. B. Lau, J. K. Y. Chan, V. S. F. Chan, M. Dalhnan, and K. S. E. Cheah (2003), Mammary gland-specific secretion of biologically active immunosuppressive agent cytotoxic-T-Iymphocyte antigen 4 human immunoglobulin fusion protein (CTLA4-IgG) in milk by transgenesis, J. Immunol. 277, 171-183 https://doi.org/10.1016/S0022-1759(03)00071-1
  14. Jeanne G. D. and A. T. Laurence (1994), New biologic immunosuppressive agents in transplantation, Curr. Opin. Nephrol. 3, 596-601 https://doi.org/10.1097/00041552-199411000-00006
  15. David, E. and S. Herb (2000), Immunological disorders, Text Book of Medicine. 4, 79-104
  16. Terashima M., Y. Ejiri, N. Hashikawa, and H. Yoshida (2001), Vtilization of an alternative carbon source for efficient production of human $a_1$-antitrypsin by genetically engineered rice cell culture, Biotechnol. Prog. 17, 403-406 https://doi.org/10.1021/bp010024p
  17. Yu, S. M., Y. C. Lee, S. C. Fang, M. T. Chan, S. F. Hwa, and L. F. Liu (1996), Sugars act as signal molecules and osmotica to regulate the expression of a-amylase genes and metabolic activities in gerntinating cereal grains, Plant Mol. BioI. 30, 1277-1289 https://doi.org/10.1007/BF00019558
  18. Battaglino, R. A., M. Huergo, A. M. R. Pilosof, and G. B. Bartholomai (1991), Culture requirements for the production of protease by Aspergillus oryzae in solid state fermentation, Appl. Microbiol. Biotechnol. 35, 292-296
  19. Bakos, A., M. Fari, O. Toldi, and M. Lados (1995), Plant regeneration from seeding-derived callus of dodder, Plant Sci. 109, 95-101 https://doi.org/10.1016/0168-9452(95)04152-K
  20. AL-Khayri, J. M. and A. M. Bahrany (2002), Callus growth and proline accumulation in response to sorbitol and sucrose-induced osmotic stress in rice, BioI. Planta 45(4), 609-611 https://doi.org/10.1023/A:1022380827034
  21. Kadota, M., K. hnizu, and T. Hiano (2001), Double-phase in vitro culture using sorbitol increases shoot proliferation and reduces hyperhydricity in Japanese pear, Scientia Horticulturae 89, 207-215 https://doi.org/10.1016/S0304-4238(00)00234-X
  22. Sajc, L., D. Grubisic, and G. Vunjak-Novakovic (2000), Bioreactors for plant engineering: an outlook for further research, Biochem. Eng. J. 4, 89-99 https://doi.org/10.1016/S1369-703X(99)00035-2
  23. Terashima M., N. Hashikawa, M. Hattori, and H. Yoshida (2002), Growth charateristic of rice cell genentically modified for recombinant human ai-antitrypsin production, Biochem. Eng. J. 12, 155-160 https://doi.org/10.1016/S1369-703X(02)00064-5