Tracing Metabolite Footsteps of Escherichia coli Along the Time Course of Recombinant Protein Expression by Two-Dimensional NMR Spectroscopy

  • Chae, Young Kee ;
  • Kim, Seol Hyun ;
  • Ellinger, James J. ;
  • Markley, John L.
  • Received : 2012.08.14
  • Accepted : 2012.09.13
  • Published : 2012.12.20


The recombinant expression of proteins has been the method of choice to meet the demands from proteomics and structural genomics studies. Despite its successful production of many heterologous proteins, Escherichia coli failed to produce many other proteins in their native forms. This may be related to the fact that the stresses resulting from the overproduction interfere with cellular processes. To better understand the physiological change during the overproduction phase, we profiled the metabolites along the time course of the recombinant protein expression. We identified 32 metabolites collected from different time points in the protein production phase. The stress induced by protein production can be characterized by (A) the increased usage of aspartic acid, choline, glycerol, and N-acetyllysine; and (B) the accumulation of adenosine, alanine, oxidized glutathione, glycine, N-acetylputrescine, and uracil. We envision that this work can be used to create a strategy for the production of usable proteins in large quantities.


Metabolite profiling;Recombinant protein;Overexpression;NMR


  1. Baneyx, F. Curr. Opin. Biotech. 1999, 10, 411.
  2. Oganesyan, N.; Ankoudinova, I.; Kim, S. H.; Kim, R. Protein Expr. Purif. 2007, 52, 280.
  3. Tegel, H.; Steen, J.; Konrad, A.; Nikdin, H.; Pettersson, K.; Stenvall,M.; Tourle, S.; Wrethagen, U.; Xu, L.; Yderland, L.; Uhlen, M.; Hober, S.; Ottosson, J. Biotechnol. J. 2009, 4, 51.
  4. Prinz, W. A.; Aslund, F.; Holmgren, A.; Beckwith, J. J. Biol. Chem. 1997, 272, 15661.
  5. Guzman, L. M.; Belin, D.; Carson, M. J.; Beckwith, J. J. Bacteriol. 1995, 177, 4121.
  6. Dyson, M. R.; Shadbolt, S. P.; Vincent, K. J.; Perera, R. L.; McCafferty, J. BMC biotechnology 2004, 4, 32.
  7. Niiranen, L.; Espelid, S.; Karlsen, C. R.; Mustonen, M.; Paulsen, S. M.; Heikinheimo, P.; Willassen, N. P. Protein Expr. Purif. 2007, 52, 210.
  8. Brown, B. L.; Hadley, M.; Page, R. Protein Expr. Purif. 2008, 62, 9.
  9. De Marco, V.; Stier, G.; Blandin, S.; de Marco, A. Biochem. Biophys. Res. Commun. 2004, 322, 766.
  10. Zhang, Y. B.; Howitt, J.; McCorkle, S.; Lawrence, P.; Springer, K.; Freimuth, P. Protein Expr. Purif. 2004, 36, 207.
  11. Malakhov, M. P.; Mattern, M. R.; Malakhova, O. A.; Drinker, M.; Weeks, S. D.; Butt, T. R. J. Struct. Funct. Genomics 2004, 5, 75.
  12. Volonte, F.; Marinelli, F.; Gastaldo, L.; Sacchi, S.; Pilone, M. S.; Pollegioni, L.; Molla, G. Protein Expr. Purif. 2008, 61, 131.
  13. Bessette, P. H.; Aslund, F.; Beckwith, J.; Georgiou, G. Proc. Natl. Acad Sci. USA 1999, 96, 13703.
  14. Brown, B. L.; Grigoriu, S.; Kim, Y.; Arruda, J. M.; Davenport, A.; Wood, T. K.; Peti, W.; Page, R. PLoS Pathogens 2009, 5, e1000706.
  15. Wang, Y. H.; Ayrapetov, M. K.; Lin, X.; Sun, G. Biochem. Biophys. Res. Commun. 2006, 346, 606.
  16. Chen, Y.; Song, J.; Sui, S. F.; Wang, D. N. Protein Expr. Purif. 2003, 32, 221.
  17. Sahu, S. K.; Rajasekharan, A.; Gummadi, S. N. Biotechnol. Lett. 2009, 31, 1745.
  18. Hoffmann, F.; Rinas, U. In Physiological Stress Responses in Bioprocesses; Springer Berlin/Heidelberg: 2004; Vol. 89, p 73.
  19. Chae, Y. K.; Cho, K. S.; Chun, W.; Lee, K. Protein Pept. Lett. 2003, 10, 369.
  20. Chae, Y. K.; Moon, W. J.; Cho, J. Y. Protein Expr. Purif. 2009, 65, 267.
  21. Chae, Y. K.; Lee, W. Bull. Korean Chem. Soc. 2008, 29, 2449.
  22. Chan, E. C.; Koh, P. K.; Mal, M.; Cheah, P. Y.; Eu, K. W.; Backshall, A.; Cavill, R.; Nicholson, J. K.; Keun, H. C. J. Proteome Res. 2009, 8, 352.
  23. Lewis, I. A.; Schommer, S. C.; Hodis, B.; Robb, K. A.; Tonelli, M.; Westler, W. M.; Sussman, M. R.; Markley, J. L. Anal. Chem. 2007, 79, 9385.
  24. Lewis, I. A.; Schommer, S. C.; Markley, J. L. Magn. Reson. Chem. 2009, 47(Suppl 1), S123.
  25. Fox, B. G.; Blommel, P. G. In Current Protocols in Protein Science; John Wiley & Sons, Inc.: 2001, p Unit 5.13.
  26. Robinette, S. L.; Ajredini, R.; Rasheed, H.; Zeinomar, A.; Schroeder, F. C.; Dossey, A. T.; Edison, A. S. Anal. Chem. 2011, 83, 1649.
  27. Motta, A.; Paris, D.; Melck, D. Anal. Chem. 2010, 82, 2405.
  28. Martineau, E.; Giraudeau, P.; Tea, I.; Akoka, S. J. Pharm. Biomed. Anal. 2011, 54, 252.
  29. Lewis, I. A.; Schommer, S. C.; Hodis, B.; Robb, K. A.; Tonelli, M.; Westler, W. M.; Suissman, M. R.; Markley, J. L. Anal. Chem. 2007, 79, 9385.
  30. Tkachenko, A.; Nesterova, L.; Pshenichnov, M. Archives of Microbiology 2001, 176, 155.

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