Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Optimization of Expression Conditions for Soluble Protein by Using a Robotic System of Multi-culture Vessels

  • Ahn, Woo-Sung (Life Sciences Division, Korea Institute of Science and Technology) ;
  • Ahn, Ji-Young (Functional Proteomics Center, Korea Institute of Science and Technology) ;
  • Jung, Chan-Hun (Department of Molecular Biology, Sejong University) ;
  • Hwang, Kwang-Yeon (School of Life Sciences & Biotechnology, Korea University) ;
  • Kim, Eunice Eun-Kyeong (Life Sciences Division, Korea Institute of Science and Technology) ;
  • Kim, Joon (School of Life Sciences & Biotechnology, Korea University) ;
  • Im, Ha-Na (Department of Molecular Biology, Sejong University) ;
  • Kim, Jin-Oh (Department of Information and Control Engineering, Kwangwoon University) ;
  • Yu, Myeong-Hee (Functional Proteomics Center, Korea Institute of Science and Technology) ;
  • Lee, Cheol-Ju (Life Sciences Division, Korea Institute of Science and Technology)
  • Published : 2007.11.30
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Abstract

We have developed a robotic system for an automated parallel cell cultivation process that enables screening of induction parameters for the soluble expression of recombinant protein. The system is designed for parallelized and simultaneous cultivation of up to 24 different types of cells or a single type of cell at 24 different conditions. Twenty-four culture vessels of about 200 ml are arranged in four columns${\times}$six rows. The system is equipped with four independent thermostated waterbaths, each of which accommodates six culture vessels. A two-channel liquid handler is attached in order to distribute medium from the reservoir to the culture vessels, to transfer seed or other reagents, and to take an aliquot from the growing cells. Cells in each vessel are agitated and aerated by sparging filtered air. We tested the system by growing Escherichia coli BL21(DE3) cells harboring a plasmid for a model protein, and used it in optimizing protein expression conditions by varying the induction temperature and the inducer concentration. The results revealed the usefulness of our custom-made cell cultivation robot in screening optimal conditions for the expression of soluble proteins.

Keywords

References

  1. Baneyx, F. and G. Georgiou. 1992. Expression of proteolytically sensitive proteins in E. coli, pp. 69-108. In Ahren, T. J. and M. C. Manning (eds.), Stability of Protein Pharmaceuticals: Chemical and Physical Paths of Protein Degradation. Plenum Press, New York
  2. Baneyx, F. and M. Mujacic. 2004. Recombinant protein folding and misfolding in Escherichia coli. Nat. Biotechnol. 22: 1399-1408 https://doi.org/10.1038/nbt1029
  3. Bollag, D. M., M. D. Rozycki, and S. J. Edelstein. 1996. Protein Methods, 2nd Ed. Wiley-Liss, Inc., New York
  4. Braun, P. and J. LaBaer. 2003. High throughput protein production for functional proteomics. Trends Biotechnol. 21: 383-388 https://doi.org/10.1016/S0167-7799(03)00189-6
  5. Burley, S. K. 2000. An overview of structural genomics. Nat. Struct. Biol. 7 Suppl: 932-934 https://doi.org/10.1038/80697
  6. Cha, K.-H., M.-D. Kim, T.-H. Lee, H.-K. Lim, K.-H. Jung, and J.-H. Seo. 2006. Coexpression of protein disulfide isomerase (PDI) enhances production of kringle fragment of human apolipoprotein(a) in recombinant Saccharomyces cerevisiae. J. Microbiol. Biotechnol. 16: 308-311
  7. Duetz, W. A., L. Ruedi, R. Hermann, K. O'Connor, J. Buchs, and B. Witholt. 2000. Methods for intense aeration, growth, storage, and replication of bacterial strains in microtiter plates. Appl. Environ. Microbiol. 66: 2641-2646 https://doi.org/10.1128/AEM.66.6.2641-2646.2000
  8. Endo, Y. and T. Sawasaki. 2004. High-throughput, genome-scale protein production method based on the wheat germ cell-free expression system. J. Struct. Funct. Genomics 5: 45-57 https://doi.org/10.1023/B:JSFG.0000029208.83739.49
  9. Friehs, K. and K. F. Reardon. 1993. Parameters influencing the productivity of recombinant E. coli cultivations. Adv. Biochem. Eng. Biotechnol. 48: 53-77
  10. Galloway, C. A., M. P. Sowden, and H. C. Smith. 2003. Increasing the yield of soluble recombinant protein expressed in E. coli by induction during late log phase. BioTechniques 34: 524-526, 528, 530
  11. Gillette, W. K., D. Esposito, P. H. Frank, M. Zhou, L. R. Yu, C. Jozwik, X. Zhang, B. McGowan, D. M. Jacobowitz, H. B. Pollard, T. Hao, D. E. Hill, M. Vidal, T. P. Conrads, T. D. Veenstra, and J. L. Hartley. 2005. Pooled ORF expression technology (POET): Using proteomics to screen pools of open reading frames for protein expression. Mol. Cell. Proteomics 4: 1647-1652 https://doi.org/10.1074/mcp.M500128-MCP200
  12. Harms, P., Y. Kostov, J. A. French, M. Soliman, M. Anjanappa, A. Ram, and G. Rao. 2006. Design and performance of a 24-station high throughput microbioreactor. Biotechnol. Bioeng. 93: 6-13 https://doi.org/10.1002/bit.20742
  13. Hedren, M., A. Ballagi, L. Mortsell, G. Rajkai, P. Stenmark, C. Sturesson, and P. Nordlund. 2006. GRETA, a new multifermenter system for structural genomics and process optimization. Acta Crystallogr. D Biol. Crystallogr. 62: 1227-1231 https://doi.org/10.1107/S090744490603441X
  14. Hong, I.-P., S. Anderson, and S.-G. Choi. 2006. Evaluation of a new episomal vector based on the GAP promoter for structural genomics in Pichia pastoris. J. Microbiol. Biotechnol. 16: 1362-1368
  15. Kachel, V., G. Sindelar, and S. Grimm. 2006. Highthroughput isolation of ultra-pure plasmid DNA by a robotic system. BMC Biotechnol. 6: 9
  16. Lee, K., H.-S. Joo, Y.-H. Yang, E. Song, and B.-G. Kim. 2006. Proteomics for Streptomyces: 'Industrial proteomics' for antibiotics. J. Microbiol. Biotechnol. 16: 331-348
  17. Lesley, S. A. 2001. High-throughput proteomics: Protein expression and purification in the postgenomic world. Protein Expr. Purif. 22: 159-164 https://doi.org/10.1006/prep.2001.1465
  18. Page, R., K. Moy, E. C. Sims, J. Velasquez, B. McManus, C. Grittini, T. L. Clayton, and R. C. Stevens. 2004. Scalable high-throughput micro-expression device for recombinant proteins. BioTechniques 37: 364-370 https://doi.org/10.2144/04373BM05
  19. Park, S.-L., E.-J. Shin, S.-P. Hong, S.-J. Jeon, and S.-W. Nam. 2005. Production of soluble human granulocyte colony stimulating factor in E. coli by molecular chaperones. J. Microbiol. Biotechnol. 15: 1267-1272
  20. Puskeiler, R., K. Kaufmann, and D. Weuster-Botz. 2005. Development, parallelization, and automation of a gas-inducing milliliter-scale bioreactor for high-throughput bioprocess design (HTBD). Biotechnol. Bioeng. 89: 512-523 https://doi.org/10.1002/bit.20352
  21. Puskeiler, R., A. Kusterer, G. T. John, and D. Weuster-Botz. 2005. Miniature bioreactors for automated high-throughput bioprocess design (HTBD): Reproducibility of parallel fedbatch cultivations with Escherichia coli. Biotechnol. Appl. Biochem. 42: 227-235 https://doi.org/10.1042/BA20040197
  22. Redaelli, L., F. Zolezzi, V. Nardese, B. Bellanti, V. Wanke, and D. Carettoni. 2005. A platform for high-throughput expression of recombinant human enzymes secreted by insect cells. J. Biotechnol. 120: 59-71 https://doi.org/10.1016/j.jbiotec.2005.05.026
  23. Samorski, M., G. Muller-Newen, and J. Buchs. 2005. Quasicontinuous combined scattered light and fluorescence measurements: A novel measurement technique for shaken microtiter plates. Biotechnol. Bioeng. 92: 61-68 https://doi.org/10.1002/bit.20573
  24. Schein, C. H. and M. H. M. Noteborn. 1988. Formation of soluble recombinant proteins in Escherichia coli is favored by lower growth temperature. Bio/Technology 6: 291-294 https://doi.org/10.1038/nbt0388-291
  25. Steen, J., M. Uhlen, S. Hober, and J. Ottosson. 2006. Highthroughput protein purification using an automated set-up for high-yield affinity chromatography. Protein Expr. Purif. 46: 173-178 https://doi.org/10.1016/j.pep.2005.12.010
  26. Studier, F. W., A. H. Rosenberg, J. J. Dunn, and J. W. Dubendorff. 1990. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 185: 60-89 https://doi.org/10.1016/0076-6879(90)85008-C
  27. Swalley, S. E., J. R. Fulghum, and S. P. Chambers. 2006. Screening factors effecting a response in soluble protein expression: Formalized approach using design of experiments. Anal. Biochem. 351: 122-127 https://doi.org/10.1016/j.ab.2005.11.046
  28. Weuster-Botz, D., J. Altenbach-Rehm, and M. Arnold. 2001. Parallel substrate feeding and pH-control in shaking-flasks. Biochem. Eng. J. 7: 163-170 https://doi.org/10.1016/S1369-703X(00)00117-0
  29. Willer, M., A. J. Jermy, B. P. Young, and C. J. Stirling. 2003. Identification of novel protein-protein interactions at the cytosolic surface of the Sec63 complex in the yeast ER membrane. Yeast 20: 133-148 https://doi.org/10.1002/yea.954
  30. Winograd, E., M. A. Pulido, and M. Wasserman. 1993. Production of DNA-recombinant polypeptides by tacinducible vectors using micromolar concentrations of IPTG. BioTechniques 14: 886-890