Korean Chemical Engineering Research

The Korean Institute of Chemical Engineers (한국화학공학회)
권호 표지

Effects of Recombinant Baculovirus Infection Conditions on Production of Green Fluorescent Protein in Drosophila S2 Cells

초파리 S2 세포 시스템에서 녹색형광단백질 생산을 위한 재조합 배큘로바이러스의 감염조건들의 영향

  • Cho, Hye Sook (Division of Molecular and Life Sciences & Department of Chemical Engineering, Pohang University of Science and Technology) ;
  • Kim, Yeon Kyu (Division of Molecular and Life Sciences & Department of Chemical Engineering, Pohang University of Science and Technology) ;
  • Kim, Kyoung Ro (Division of Molecular and Life Sciences & Department of Chemical Engineering, Pohang University of Science and Technology) ;
  • Cha, Hyung Joon (Division of Molecular and Life Sciences & Department of Chemical Engineering, Pohang University of Science and Technology)
  • 조혜숙 (포항공과대학교 화학공학과, 분자생명과학부) ;
  • 김연규 (포항공과대학교 화학공학과, 분자생명과학부) ;
  • 김경로 (포항공과대학교 화학공학과, 분자생명과학부) ;
  • 차형준 (포항공과대학교 화학공학과, 분자생명과학부)
  • Received : 2005.11.21
  • Accepted : 2005.12.23
  • Published : 2006.02.28
Download PDF
⟨ Previous Next ⟩

Abstract

The baculovirus-insect Drosophila melanogaster S2 cell system combines advantages of conventional baculovirus system and non-lytic S2 cell system because baculoviruses can infect non-permissive cells such as mammalian and Drosophila S2 cells but cannot replicate themselves inside the cells. In the present work, we investigated effects of infection conditions on production of green fluorescent protein (GFP) as a target protein using this baculovirus-S2 cell system. Even though higher MOI and longer baculovirus contact time showed better GFP expression yield during the shorter period, overall protein yield could be lower during the longer period due to the relatively higher cell detachment and lysis (lower cell viability). In addition, maintaining high MOI will be not practical for large-scale cell culture. Therefore, instead of maintaining high MOI, we found that high initial cell number and concentrated (10X) baculovirus volume can confer comparable protein expression even under the moderate MOI condition. Also, we found that the post-infection time that is connected to state of cells after infection was an important factor for production yield.

원하는 유전자를 배큘로바이러스의 감염에 의하여 초파리 Drosophila melanogaster S2 세포에 도입하는 배큘로바이러스/S2 세포 발현 시스템은 강력하고 안전한 배큘로바이러스의 장점과 세포가 파괴되지 않는 S2 세포의 장점을 접목한 시스템이다. 본 연구에서는 외래 목적 단백질로 발현 모니터링이 손쉬운 녹색형광단백질(green fluorescent protein)을 발현시키는 재조합 배큘로바이러스를 이용하여 S2 세포에의 감염조건들에 대한 영향 연구를 수행하였다. 재조합 배큘로바이러스의 S2 세포에의 감염조건으로는 multiplicity of infection(MOI), 초기 세포 수, 배큘로바이러스 용액이 세포를 적시는 최소 부피, 배큘로바이러스를 첨가한 후 제거하기까지의 배양시간 그리고 배큘로바이러스 제거 후 S2 세포를 혈청이 존재하는 배지에서 키우는 배양시간을 선정하였다. MOI는 일반적으로 크게 하는 것이 높은 발현 수율을 위하여 좋은 결과를 보였으나 세포배양이 길어지는 경우 세포독성의 문제가 심각해 질 수 있고 실제적인 배양에서는 높은 MOI를 유지하는 것이 불가능하므로 사용하는 100 mm 배양접시에서 배큘로바이러스와 S2 세포가 접촉하는 동안의 변수들인 MOI를 적정의 값인 30으로, 배큘로바이러스 배양 시간을 1.5 시간으로 고정함으로써 배큘로바이러스가 숙주 세포인 S2에 대해 미치는 세포독성을 낮출 수 있었다. 또한, 배큘로바이러스 최소 부피는 배양접시에서 사용부피의 2.4％에서 가장 좋은 결과를 보였으며 배큘로바이러스 용액을 10배 농축시킴으로써 이 부피를 맞추기 위해 첨가시키는 새로운 배지의 비율을 높임으로써 결과적으로 배큘로바이러스에 의한 세포독성을 줄일 수 있었다. 이를 통하여 세포 파쇄 및 배양접시 표면에서 탈착되는 것을 막고 높은 생장 상태를 유지해서 감염 효율도 높일 수 있었다. 배큘로바이러스의 감염이 끝난 후의 관여하는 변수들인 감염 후 배양시간은 24시간에서 최대의 녹색형광단백질의 발현을 나타내었다.

Keywords

Acknowledgement

Supported by : 학술진흥재단

References

  1. Schneider, I., 'Cell Lines Derived from Late Embryonic Stages of Drosophila melanogaster,' J. Embryol. Exp. Morphol., 27, 353-365(1972)
  2. van der Straten, A., Johansen, H., Rosenberg, M. and Sweet, R. W., 'Introduction and Constitutive Expression of Gene Products in Cultured Drosophila Cells Using Hygromycin B Selection,' Curr. Methods Mol. Cell Biol., 1, 1-8(1989) https://doi.org/10.1016/S0955-0674(89)80027-4
  3. Johansen, H., van der Straten, A., Sweet, R., Otto, E., Maroni, G. and Rosenberg, M., 'Regulated Expression at High Copy Number Allows Production of a Growth-inhibitory Oncogene Product in Drosophila Schneider Cells,' Genes Div., 3, 882-889(1989) https://doi.org/10.1101/gad.3.6.882
  4. Kirkpatrick, R. B., Ganguly, S., Angelichio, M., Griego, S., Shatzman, A., Silverman, C. and Rosenberg, M., 'Heavy Chain Dimers as well as Complete Antibodies are Efficiently Formed and Secreted from Drosophila via a BiP-mediated Pathway,' J Biol Chem., 270, 19800-19805(1995) https://doi.org/10.1074/jbc.270.34.19800
  5. Li, B., Tsing, S., Kosaka, A. H., Nguyen, B., Osen, E. G., Bach, C., Chan, H. and Barnett, J., 'Expression of Human Dopamine Beta-hydroxylase in Drosophila Schneider 2 Cells,' Biochem J., 313, 57-64(1996) https://doi.org/10.1042/bj3130057
  6. Lehr, R. V., Elefante, L. C., Kikly, K. K., O'Brien, S. P. and Kirkpatrick, R. B., 'A Modified Metal-ion Affinity Chromatography Procedure for the Purification of Histidine-tagged Recombinant Proteins Expressed in Drosophila S2 Cells,' Protein Expr Purif., 19, 362-368(2000) https://doi.org/10.1006/prep.2000.1258
  7. Corchero, J. L. and Villaverde, A., 'Plasmid Maintenance in Escherichia coli Recombinant Cultures is Dramatically, Steadily, and Specifically Influenced by Features of the Encoded Proteins,' Biotechnol Bioeng., 58, 625-632(1998) https://doi.org/10.1002/(SICI)1097-0290(19980620)58:6<625::AID-BIT8>3.0.CO;2-K
  8. Dobrosotskaya, I. Y., Goldstein, J. L., Brown, M. A. and Rawson, R. B., 'Reconstitution of Sterol-regulated Endoplasmic Reticulum- to-Golgi Transport of SREBP-2 in Insect Cells by Co-expression of Mammalian SCAP and Insigs,' J Biol Chem., 278, 35837-35843 (2003) https://doi.org/10.1074/jbc.M306476200
  9. Liu, X., Constantinescu, S. N., Sun, Y., Bogan, J. S., Hirsch, D., Weinberg, R. A. and Lodish, H. F., 'Generation of Mammalian Cells Stably Expressing Multiple Genes at Predetermined Levels,' Anal Biochem., 280, 20-28(2000) https://doi.org/10.1006/abio.2000.4478
  10. Miller, M. K., 'Baculoviruses as Gene Expression Vectors,' Ann Rev Microbiol., 42, 177-199(1988) https://doi.org/10.1146/annurev.mi.42.100188.001141
  11. Cha, H. J., Pham, M. Q., Rao, G. and Bentley, W. E., 'Expression of Green Fluorescent Protein in Insect Larvae and its Application for Foreign Protein Production,' Biotechnol Bioeng., 56, 239-247(1997) https://doi.org/10.1002/(SICI)1097-0290(19971105)56:3<239::AID-BIT1>3.0.CO;2-K
  12. Cha, H. J., Dalal, N. G., Vakharia, V. N. and Bentley, W. E., 'Expression and Purification of Human Interleukin-2 Simplified as a Fusion with Green Fluorescent Protein in Suspended Insect Sf-9 Cells,' J Biotechnol., 69, 9-17(1999) https://doi.org/10.1016/S0168-1656(98)00190-4
  13. O'Reilly, D. R. and Miller, L. K., 'Expression and Complex Formation of Simian Virus 40 Large T Antigen and Mouse p53 in Insect Cells,' J Virol., 62, 3109-3119(1988)
  14. Hu, Y. C. and Bentley, W. E., 'Effect of MOI Ratio on the Composition and Yield of Chimeric Infectious Bursal Disease Virus-like Particles by Baculovirus Co-infection: Deterministic Predictions and Experimental Results,' Biotechnol Bioeng., 75, 104-119(2001) https://doi.org/10.1002/bit.1170
  15. Carbonell, L. F., Klowden, M. J. and Miller, L. K., 'Baculoviursmediated Expression of Bacterial Genes in Dipteran and Mammalian Cells,' J Virol., 56, 153-160(1985)
  16. Morris, T. D. and Miller, L. K., 'Promoter Influence on Baculovirus- mediated Gene Expression in Permissive and Nonpermissive Insect Cell Lines,' J Virol., 66, 7397-7405(1992)
  17. Lee, D. F., Chen, C. C., Hsu, T. A. and Juang, J. L., 'A Baculovirus Superinfection System: Efficient Vehicle for Gene Transfer into Drosophila S2 Cells,' J Virol., 74, 11873-11880(2000) https://doi.org/10.1128/JVI.74.24.11873-11880.2000
  18. Cho, H. S., Kim, Y. K. and Cha, H. J., 'Expression of Double Foreign Protein Types Following Recombinant Baculovirus Infection of Stably Transfected Drosophila S2 Cells,' Enzyme and Microbial Technology., 35, 525-531(2004) https://doi.org/10.1016/j.enzmictec.2004.08.004
  19. Joseph, M., 'Gene Expression System,' Academic press., 331- 372(1999)
  20. Hu, Y. C., Tsai, C. T., Chang, Y. J. and Huang, J.U., 'Enhancement and Prolongation of Baculovirus-mediated Expression in Mammalian Cells: Focuses on Strategic Infection and Feeding,' Biotechnol. Prog., 19, 373-379(2003) https://doi.org/10.1021/bp025609d
  21. Friedrich, A., Erika, S., Iain, B. H. W. and Leopold, M., 'Insect Cells as Hosts for the Expression of Recombinant Glycoproteins,' Glycoconjugate jornal., 16, 109-123(1999) https://doi.org/10.1023/A:1026488408951
  22. Duisit, S., Saleun, S., Douthe, S., Barsoum, J., Chadeuf, G. and Moullier, P., 'Baculovirus Vetor Requires Electrostatic Interactions Including Heparin Sulfate for Efficient Gene Transfer in Mammalian Cells,' The J. of gene medicine., 1, 93-102(1999) https://doi.org/10.1002/(SICI)1521-2254(199903/04)1:2<93::AID-JGM19>3.0.CO;2-1