KSBB Journal

  • 제21권4호
  • /
  • Pages.236-240
  • /
  • 2006
  • /
  • 1225-7117(pISSN)
  • /
  • 2288-8268(eISSN)
한국생물공학회 (The Korean Society for Biotechnology and Bioengineering)
권호 표지

발현유도에 의한 알칼리 소비속도의 감소가 재조합 단백질 생산에 미치는 영향

The Dependency of the Expression Level of Recombinant Protein by the Drop of Alkali Consumption Rate after Induction

  • 허원 (강원대학교 공과대학 생물공학과)
  • Hur, Won (Department of Bioengineering and Technology, College of Engineering, Kangwon National University)
  • 발행 : 2006.08.30
PDF 다운로드
⟨ 이전 논문 다음 논문 ⟩

초록

대장균을 회분배양하면서 알칼리소비속도를 온라인으로 모니터하고, 대수증식기에 IPTG로 재조합 단백질의 발현을 유도시키면 알칼리소비속도가 급격하게 감소하는 것을 확인 할 수 있다. 회분배양을 서로 다른 조건에서 7회 실시하고 ${\beta}$-galactosidase의 발현량과 발현유도 직후 알칼리 소비속도의 감소를 비교하여 알칼리소비의 감소가 클수록 발현량이 증가한다는 것을 알 수 있었다. 그러나 IPTG를 재투입하여도 발현량은 증가하지 않았고 배지를 추가로 공급하면 발현량이 증가한다는 것을 확인하였다. 이와 같은 결과로부터 외래단백질의 발현속도는 초기의 IPTG 투입시 결정되고 이후의 재조합단백질의 생산은 배지의 공급에 의하여 제한된다고 판단할 수 있다. 이와 같은 알칼리 소비속도는 Casamino acid를 질소원으로 사용할 경우 관찰 되었으나 Yeast extract를 유일한 탄소원으로 사용할 경우에는 관찰되지 않았다.

IPTG induction caused a sudden drop of alkali consumption rate during cultivation of a recombinant E. coli with ${\beta}$-galactosidase structural gene under T7 promoter on a plasmid. A series of batch cultivations showed the positive correlation of the decrease of alkali consumption and the level of expression. However, repeated IPTG induction did not cause any variation of alkali consumption rate. Supplementation of medium even at stationary phase enhanced the level of ${\beta}$-galactosidase expression. These results suggests that the drop of alkali consumption rate by IPTG induction represents the rate of expression.

키워드

참고문헌

  1. Ellis, R. J. (2001), Macromolecular crowing: obvious but under appreciated, Trends Biochem. Sci. 26, 597-604 https://doi.org/10.1016/S0968-0004(01)01938-7
  2. Bentley, W. E., N. Mirjalili, D. C. Anderson, R. H. Davis, and D. S. Kompala (1990), Plasmid encoded protein: the principal factor of 'metabolic burden' associated with recombinant bacteria, Biotechnol. Bioeng. 35, 668-681 https://doi.org/10.1002/bit.260350704
  3. Glick, B. R. (1995), Metabolic load and heterologous gene expression, Biotechnol Adv. 13, 247-261 https://doi.org/10.1016/0734-9750(95)00004-A
  4. Kosinski, M. J., U. Rinas, and J. E. Bailey (1992), Isopropyl-$\beta$ -D-thiogalactopyranoside influences the metabolism of Escherichia coli, Appl. Environ. Microbiol. 36, 782-784
  5. Lilie, H., E. Schwarz, and R. Rudolph (1998), Advances in refolding of proteins produced in E. coli, Curr. Opin. Biotechnol. 9, 497-501 https://doi.org/10.1016/S0958-1669(98)80035-9
  6. Gill, R. T., J. J. Valdes, and W. E. Bentley (1999), Reverse transcription PCR differential display analysis of Escherichia coli global gene regulation in response to heat shock, Appl. Environ. Microb. 65, 5386-5393
  7. Jurgen, B., H. Y. Lin, S. Riemschneider, C. Scharf, P. Neubauer, R. Schmidt, M. Hecker, and T. Schweder (2000), Monitoring of genes that respond to over production of an insoluble recombinant protein in Escherichia coli glucose-limited fed-batch fermentations, Biotechnol. Bioeng. 70, 217-224 https://doi.org/10.1002/1097-0290(20001020)70:2<217::AID-BIT11>3.0.CO;2-W
  8. Zheng, M., X. Wang, L. J. Templeton, D. R. Smulski, R. A. LaRossa, and G. Storz (2001), DNA microarray-mediated transcriptional profiling of the Escherichia coli response to hydrogen peroxide, J. Bacteriol. 183, 4562-4570 https://doi.org/10.1128/JB.183.15.4562-4570.2001
  9. Cha, H. J., R. Srivastava, V. M. Vakharia, G. G. Rao, and W. E. Bentry (1999), Green fluorescent protein as a noninvasive stress probe in resting Escherichia coli cells, Appl. Environ. Microb. 65, 409-414
  10. Vostiar, I., J. Tkac, and C. F. Mandenius (2004), Off-line monitoring of bacterial stress response during recombinant protein production using an optical biosensor, J. Biotechnol. 11, 191-201
  11. Hur, W. and Y. K. Chung (2005) On-line monitoring of IPTG induction for recombinant protein production using an automatic pH control signal, Biotechnol. Bioprocess. Eng. 10, 304-308 https://doi.org/10.1007/BF02931846
  12. Schügerl, K. (1991), Common Instruments for Process Analysis and Control, In Biotechnology Vol. 4, H. J. Rehm, G. Reed, A. Puhler and P. Stadler Eds., p6-25, 1991, VCH Publishers Inc., New York
  13. Suzuki T., T. Yamane, and S. Shimizu (1990), Phenomenological background and some preliminary trials of automated substrate supply in pH-stat model fed-batch culture using a set point of high limit, J. Ferment. Bioengin. 69, 292-297 https://doi.org/10.1016/0922-338X(90)90108-9
  14. Chung Y. K. and W. Hur (2000), A new method of measuring of buffer capacity and alkali consumption rate of a fermentation process, J. Bioscience. Bioeng. 90, 580-582 https://doi.org/10.1016/S1389-1723(01)80047-5
  15. Miller, G. L. (1959), Use of dinitrosalicylic acid reagent for determination of reduction sugar, Anal. Chem. 31, 426 https://doi.org/10.1021/ac60147a030
  16. Miller, J. H. (1972), Experiments in molecular genetics, 2rd ed., p128, Cold Spring Harbor, New York
  17. Zaslaver, A., A. E. Mayo, R. Rosenberg, P. Bashkin, H. Sberro, M. Tsalyuk, M. G. Surette, and U. Alon (2004), Just-in-time transcription program in metabolic pathways, Nature Genetics 36, 486-491 https://doi.org/10.1038/ng1348
  18. Studier, F. and B. Moffatt (1986), Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes, J. Mol. Biol. 189, 113-130 https://doi.org/10.1016/0022-2836(86)90385-2
  19. Albano, C. R., L. Randers-Eichhorn, W. E. Bentley, and G. Rao (1998), Green fluorescent protein as a real time quantitative reporter of heterologous protein production, Biotechnol. Prog. 14, 350-354
  20. Jones J. J., A. M. Bridges, A. P. Fosberry, S. Gardner, R. R. Lowers, R. R. Newby, P. J. James, R. M. Hall, and O. Jenkins (2004), Potential of real-time measurement of GFP-fusion proteins, J. Biotechnol. 109, 201-211 https://doi.org/10.1016/j.jbiotec.2003.10.039