생명과학회지 (Journal of Life Science)

한국생명과학회 (Korean Society of Life Science)
권호 표지
DOI QR코드

DOI QR Code

형질전환된 벼세포배양에서 green fluorescent protein (GFP) 생산

Production of Green Fluorescent Protein (GFP) from Transgenic Rice Cell Suspension Culture

  • 이재화 (신라대학교 의생명과학대 제약공학과)
  • Lee, Jae-Hwa (Department of Pharmaceutical Engineering, College of Medical Life Science, Silla University)
  • 발행 : 2007.02.28
PDF 다운로드
⟨ 이전 논문 다음 논문 ⟩

초록

형광단백질 (green fluorescent protein, GFP)은 생물공정을 살피데 지표 단백질로 유용하게 사용이 된다. 본 연구에서는 쌀세포에서 외래 단백질의 발현양상을 관찰하기 위해서, 표지 단백질로 GFP를 형질전환 후 이것에서 유도된 현탁세포에서 GFP의 발현 양상을 관찰하였다. 형질전환시 GFP의 발현을 위한 프로모터로 RAmysE를 사용하였으며 이것은 배양액 중에서 당이 고갈되었을 때 강력히 작동된다. 그래서 본 연구에서는 배양액 중에 다양한 슈크로오스 농도로 쌀세포를 배양하여 세포의 성장양태 및 GFP의 발현양에 미치는 영향을 관찰한 결과 세포의 성장은 12%의 당농도에서 7.06g/L로 최적이였으며 GFP는 당을 가장 적게 사용한 3%에서 최적임을 알 수가 있었다. 이것은 세포의 성장과 GFP의 생산에 사용된 당이 반대로 영향을 미침을 알 수가 있었으며 향후 최적의 대량배양을 위해서는 세포의 성장과 산물의 생산시기를 분리한 이단계 배양법이 필요함을 암시한다.

Green fluorescent protein (GFP) is an attractive reporter for bioprocess monitoring. A fluorescence-based method was developed to quantify GFP levels in transgenic plants and protein extracts. In this study, GFP was produced and secreted from suspension cells derived from transgenic rice. The RAmy3E promoter placed before the GFP gene controlled by sugars such as sucrose. The effects of sucrose concentration on the secretion of GFP and total protein into the medium were investigated in batch suspension culture. It was possible, therefore, to induce the expression of the GFP by removing sucrose from the cultured media or by allowing the rice suspension cells to deplete sucrose catabolically. The dry cell weight (7.06 g/L) and GFP level were detected as highest at 12%, 3% sucrose after 20 day culture, respectively. However secreted GFP fluorescence at the other sucrose concentrations (6%, 12%, 18% and 24%) were a little amount in media.

키워드

참고문헌

  1. Cha, H. J., C. F. Wu, J. J. Valdes, G. Rao and W. E. Bentley. 2000. Observations of green fluorescent protein as a fusion partner in genetically engineered Escherichia coli: Monitoring protein expression and solubility. Biotechnol. Bioeng. 67, 565-574 https://doi.org/10.1002/(SICI)1097-0290(20000305)67:5<565::AID-BIT7>3.0.CO;2-P
  2. Cha, H. J., N. G. Dalal, M. Q. Pham, V. N. Vakharia and W. E. Bentley. 1999. Insect larval expression process is optimized by generating fusions with green fluorescent protein. Biotechnol. Bioeng. 65, 316-324 https://doi.org/10.1002/(SICI)1097-0290(19991105)65:3<316::AID-BIT9>3.0.CO;2-X
  3. Choi, H. K., J. S. Son, G. H. Na, S. S. Hong, Y. S. Park and J. Y. Song. 2002. Mass production of Paclitaxel by plant cell culture. Korean J Plant Biotechnol. 29, 59-62 https://doi.org/10.5010/JPB.2002.29.1.059
  4. Doran, P. M. 2000. Foreign protein production in plant tissue cultures. Curr. Opin. in Biotechnol. 11, 199-204 https://doi.org/10.1016/S0958-1669(00)00086-0
  5. Huang, N., J. Chandler, B. R. Thomas, N. Koizumi and R. L. Rodriguez. 1993. Metabolic regulation of alpha-amylase gene expression in transgenic cell cultures of rice (Oryza sativa L.). Plant Mol. Biol. 23, 737-747 https://doi.org/10.1007/BF00021529
  6. Kim, S. M., J. S. Park, Y. Lee, G. W. Lee and D. J. Kim. 1998. Changes of plant cell size index by culture conditions, Kor. J. Biotechnol. Bioeng. 13, 438-443
  7. LaCount, W., G. An and J. M. Lee. 1997. The effect of polyvinylpyrokidone (PVP) on the heavy chain monoclonal antibody production from plant suspension cultures. Biotechnol. Lett. 19, 93-96 https://doi.org/10.1023/A:1018383524389
  8. Liu, S., R. C. Bugos, N. Dharmasiri and W. W. Su. 2001 . Green fluorescent protein as a secretory reporter and a tool for process optimization in transgenic plant cell cultures. J. Biotechnol. 87, 1-16 https://doi.org/10.1016/S0168-1656(00)00421-1
  9. Matsumoto, S., M. Ikura, M. Ueda and R. Sasaki. 1995. Characterization of a human glycoprotein (erythroprotein) produced in cultured tobacco cells. Plant Mol. Biol. 27, 1163-1172 https://doi.org/10.1007/BF00020889
  10. Miele, L., 1997. Plants as bioreactors for biopharmaceuticals: regulatory considerations. Trends Biotechnol. 15, 45-50 https://doi.org/10.1016/S0167-7799(97)84202-3
  11. Nakano, H., R. Okumura, C. Goto and T. Yamane. 2002. In vitro combinatorial mutagenesis of the 65th and 222nd positions of the green fluorescent protein of Aequarea victoria. Biotechnol. Bioprocess Eng. 7, 311-315 https://doi.org/10.1007/BF02932841
  12. Ohga, M., M. Ogura, M. Matasumura and P. C. Wang. 2002. Construction of glomerular epithelial cells expressing both immune tolerance and GFP genes and application to cell therapy by cell transplantation, Biotechnol. Bioprocess Eng. 7, 303-310 https://doi.org/10.1007/BF02932840
  13. Payne, G., V. Bringi, C. Prince and M. Shuler. Suspension culture in plant cell and tissue culture in liquid systems, Oxford University Press, New York 146-176
  14. Richards, H. A., M. D. Halfhill, R. J. Millwood and C. N. Stewart. 2003. Quantitative GFP fluorescence as an indicator of recombinant protein synthesis in transgenic plants, Plant Cell Rep. 22, 117-121 https://doi.org/10.1007/s00299-003-0638-1
  15. 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-marcrophage colony stimulation factor in transgenic rice cell suspension culture. Biotechnol. Bioeng. 82, 778-783 https://doi.org/10.1002/bit.10635
  16. Stoscheck, C. M. 1990. Quantitation of Protein, Methods in Enzymology 182, 50-68 https://doi.org/10.1016/0076-6879(90)82008-P
  17. Subramanian, S. and F. Srienc. 1996. Quantitative analysis of transient gene expression in mammalian cells using the green fluorescent protein, J. Biotechnol. 49, 137 https://doi.org/10.1016/0168-1656(96)01536-2
  18. Terashima, M., Y. Ejiri, N. Hashikawa and H. Yoshida. 2001. Utilization of a alternative carbon source for efficient production of human $a_1$-antltrypsin by genetically engineered rice cell culture. Biotechnol. Prog. 17, 403-406 https://doi.org/10.1021/bp010024p
  19. Terashima, M., Y. Murai, M. Kawamura, S. Nakanishi, T. Stoltz, L. Chen, W. Drohan, R. L. Rodriguez and S. Katoh. 1999. Production of functional human ${\alpha}\;_1$-antitrypsin by plant cell culture, Appl. Microbiol. Biotechnol. 52, 516-523 https://doi.org/10.1007/s002530051554
  20. Yu, 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 ${\alpha}$-amylase genes and metabolic activities in germinating cereal grains. Plant Mol. Biol. 30, 1277-1289 https://doi.org/10.1007/BF00019558

피인용 문헌

  1. Optimized Germination Conditions and Human p53 Expression of Rice Embryo vol.25, pp.2, 2015, https://doi.org/10.5352/JLS.2015.25.2.158