Skip to main content
SpringerLink
Log in

Simulation of large-scale production of a soluble recombinant protein expressed in Escherichia coli using an intein-mediated purification system

  • Published:
Applied Biochemistry and Biotechnology Aims and scope Submit manuscript

Abstract

Inteins are self-cleavalbe proteins that under reducing conditions can be cleaved from a recombinant target protein. Industrially, an intein-based system could potentially reduce production costs of recombinant proteins by facilitating a highly selective affinity purification using an inexpensive substrate such as chitin. In this study, SuperPro® Designer was used to simulate the large-scale recovery of a soluble recombinant protein expressed in Escherichia coli using an intein-mediated purification process based on the commercially available IMPACT® system. The intein process was also compared with a conventional process simulated by SuperPro. The intein purification process initially simulated was significantly more expensive than the conventional process, primarily owing to the properties of the chitin resin and high reducing-agent (dithiothreitol [DTT]) raw material cost. The intein process was sensitive to the chitin resin binding capacity, cleavage efficiency of the intein fusion protein, the size of the target protein relative to the intein tag, and DTT costs. An optimized intein purification process considerably reduced costs by simulating an improved chitin resin and alternative reduced agents. Thus, to realize the full potential of intein purification processes, research is needed to improve the properties of chitin resin and to find alternative, inexpensive raw materials.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Petrides, D. P., Koulouris, A., and Lagonikos, P. T. (2002), Pharm. Eng. 22, 56–65.

    Google Scholar 

  2. Rouf, S. A., Douglas, P. L., Moo-Yoong, M., and Scharer, J. M. (2001), Biochem. Eng. J. 8, 229–234.

    Article  CAS  Google Scholar 

  3. Shanklin, T., Roper, K., Yegeswaran, P. K., and Marten, M. R. (2001), Biotechnol. Bioeng. 72, 483–489.

    Article  PubMed  CAS  Google Scholar 

  4. Chong, S. R., Williams, K. S., Wotkowicz, C., and Xu, M. Q. (1998), J. Biol. Chem. 273, 10,567–10,577.

    Article  CAS  Google Scholar 

  5. Chong, S. R., Montello, G. E., Zhang, A. H., Cantor, E. J., Liao, W., Xu, M. Q., and Benner, J. (1998), Nucleic Acids Res. 26, 5109–5115.

    Article  PubMed  CAS  Google Scholar 

  6. Hong, S. H., Toyama, M., Maret, W., and Murooka, Y. (2001), Protein Express. Pur. 21, 243–250.

    Article  CAS  Google Scholar 

  7. Wu, C., Seitz, P. K., and Falzon, M. (2000), Mol. Cell. Endocrinol. 70, 163–174.

    Article  Google Scholar 

  8. Chong, S. H., Mersha, F. B., Comb, D. G., et al. (1997), Gene 192, 271–281.

    Article  PubMed  CAS  Google Scholar 

  9. Southworth, M., Amaya, K., Evans, T., Xu, M., and Perler, F. (1999), Biotechniques 27, 110–120.

    PubMed  CAS  Google Scholar 

  10. Mathys, S., Evans, T., Chute, I., Wu, H., Chong, S., Benner, J., Liu, X., and Xu, M. (1999), Gene 231, 1–13.

    Article  PubMed  CAS  Google Scholar 

  11. Zhang, A. H., Gonzalez, S. M., Cantor, E. J., and Chong, S. R. (2001), Gene 275, 241–252.

    Article  PubMed  CAS  Google Scholar 

  12. Wood, D. W., Wu, W., Belfort, G., Derbyshire, V., and Belfort, M. (1999), Nat. Biotechnol. 17, 889–892.

    Article  PubMed  CAS  Google Scholar 

  13. Wood, D. W., Derbyshire, V., Wu, W., Chartrain, M., Belfort, M., and Belfort, G. (2000), Biotech. Prog. 16, 1055–1063.

    Article  CAS  Google Scholar 

  14. Thomson, C. A. and Ananthanarayanan, V. S. (2001), Prot. Expr. Purif. 23, 8–13.

    Article  CAS  Google Scholar 

  15. Grzybowska, B., Szweda, P., and Synowiecki, J. (2004), Mol. Biotechnol. 26, 101–109.

    Article  PubMed  CAS  Google Scholar 

  16. Carrio, M. M. and Villaverde, A. (2002), J. Biotechnol. 96, 3–12.

    Article  PubMed  CAS  Google Scholar 

  17. Jordan, G. L. and Harcum, S. W. (2002), J. Ind. Microbiol. Biotechnol. 28, 74–80.

    Article  PubMed  CAS  Google Scholar 

  18. Ernst, S., Garro, O. A., Winkler, S., Venkatarnaman, G., Langer, R., Cooney, C. L., and Sasisekharan, R. (1997), Biotechnol. Bioeng. 53, 575–582.

    Article  CAS  Google Scholar 

  19. Ladisch, M. (2001), Bioseparations Engineering: Principles, Practice, and Economics, 1st ed., John Wiley & Sons, Indianapolis.

    Google Scholar 

  20. Protein Purification—Handbook. (2001), Amersham Biosciences AB, Uppsala, Sweden.

  21. Sharma, S. S., Zhang, A., Wang, H., Harcum, S. W., and Chong, S. (2003), Biotech. Prog. 19, 1085–1090.

    Article  CAS  Google Scholar 

  22. Datar, R. V., Cartwright, T., and Rosen, C. G. (1993), Bio/Technology 11, 349–357.

    Article  PubMed  CAS  Google Scholar 

  23. Evangelista, R. L., Kusnadi, A. R., Howard, J. A., and Nikolov, N. L. 1998), Biotech. Prog. 14, 607–614.

    Article  CAS  Google Scholar 

  24. Petrides, D. P., Sapidou, E., and Calandranis, J. (1995), Bioitechnol. Bioeng. 48, 529–541.

    Article  CAS  Google Scholar 

  25. Brierley, R. A., Abrams, J. N., Hanson, J. M., and Maslanka, F. C. (2001), US patent 6,207,806 B1.

  26. Koths, K. Thomson, J., Kunitani, M., Wilson, K., and Hanisch, W. (1986), US patent 4,569,790.

  27. Stern, A. S. (1998), US patent 5,831,022.

  28. Cantor, E. J. and Chong, S. R. (2001), Prot. Expr. Purif. 22, 135–140.

    Article  CAS  Google Scholar 

  29. Harrison, R. G., Todd, P., Rudge, S. R., and Petrides, D. P. (2003), Bioseparations Science and Engieering, Oxford University Press, New York.

    Google Scholar 

  30. Peters, M. S. and Timmerhaus, K. D. (1991), Plant Design and Economics for Chemical Engineers, 4th ed., McGraw-Hill, New York.

    Google Scholar 

  31. Garnett, D. I. and Patience, G. S. (1993), Chem. Eng. Prog. 89, 76–78.

    Google Scholar 

  32. Roberts, G. A. F. and Taylor, K. E. (1988), in Chitin and Chitosan—Sources, Chemistry, Biochemistry, Physical Properties and Applications, Skjak-Braek, G., Anthonsen, T., and Sandford, P., eds., Elsevier Applied Science, London, pp. 577–583.

    Google Scholar 

  33. Seo, H. and Kinemura, Y. (1988), in Chitin and Chitosan—Sources, Chemistry, Biochemistry, Physical Properties and Applications, Skjak-Braek, G., Anthonsen, T., and Sandford, P., eds., Elsevier Applied Science, London, pp. 585–588.

    Google Scholar 

  34. Ma, J. and Cooney, C. L. (2004), Biotech. Prog. 20, 269–276.

    Article  CAS  Google Scholar 

  35. Barker, A. F., Siemsen, F., Pasley, D., D'Silva, R., and Buist, A. S. (1994), Chest 105, 1406–1410.

    PubMed  CAS  Google Scholar 

  36. deSerres, F. J. (2002), Chest 122, 1818–1829.

    Article  Google Scholar 

  37. Lyddiatt, A. (2002), Curr. Opin. Biotechnol. 13, 95–103.

    Article  PubMed  CAS  Google Scholar 

  38. Persson, J., Andersen, D. C., and Lester, P. M. (2005), Biotechnol. Bioeng. 90, 442–451.

    Article  PubMed  CAS  Google Scholar 

  39. Willoughby, N., Martin, P., and Tichener-Hooker, N. (2004), Biotechnol. Bioeng. 87, 641–647.

    Article  PubMed  CAS  Google Scholar 

  40. Ling, T. C., Lyddiatt, A., Carmichael, I., Purdom, G., Hathi, P., and Levison, P. R. (2003), J. Chromatogr. A 989, 109–118.

    Article  PubMed  CAS  Google Scholar 

  41. Fahrner, R. L., Blank, G. S., and Zapata, G. A. (1999), J. Biotechnol. 75, 273–280.

    Article  PubMed  CAS  Google Scholar 

  42. Anspach, F. B., Curbelo, D., Hartmann, R., Garke, G., and Deckwer, W.-D. (1999), J. Chromatogr. A 865, 129–144.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Chemical Engineering, Clemson University, 127 Earle Hall, 29634-0905, Clemson, SC

    Shamik S. Sharma

  2. New England Biolabs, 32 Tozer Road, 01915, Beverly, MA

    Shaorong Chong

  3. Department of Bioengineering, 401 Rhodes Engineering Research Center, Clemson University, 29634-0905, Clemson, SC

    Sarah W. Harcum

Authors
  1. Shamik S. Sharma
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Shaorong Chong
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sarah W. Harcum
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Sarah W. Harcum.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Sharma, S.S., Chong, S. & Harcum, S.W. Simulation of large-scale production of a soluble recombinant protein expressed in Escherichia coli using an intein-mediated purification system. Appl Biochem Biotechnol 126, 93–117 (2005). https://doi.org/10.1385/ABAB:126:2:093

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1385/ABAB:126:2:093

Index Entries

Search

Navigation