Skip to main content
SpringerLink
Log in

Optimizing Periplasmic Expression in Escherichia coli for the Production of Recombinant Proteins Tagged with the Small Metal-Binding Protein SmbP

  • Original paper
  • Published:
Molecular Biotechnology Aims and scope Submit manuscript

Abstract

We have previously shown that the small metal-binding protein (SmbP) extracted from the gram-negative bacterium Nitrosomonas europaea can be employed as a fusion protein for the expression and purification of recombinant proteins in Escherichia coli. With the goal of increasing the amounts of SmbP-tagged proteins produced in the E. coli periplasm, we replaced the native SmbP signal peptide with three different signal sequences: two were from the proteins CusF and PelB, for transport via the Sec pathway, and one was the signal peptide from TorA, for transport via the Tat pathway. Expression of SmbP-tagged Red Fluorescent Protein (RFP) using these three alternative signal peptides individually showed a considerable increase in protein levels in the periplasm of E. coli as compared to its level using the SmbP signal sequence. Therefore, for routine periplasmic expression and purification of recombinant proteins in E. coli, we highly recommend the use of the fusion proteins PelB-SmbP or CusF-SmbP, since these signal sequences increase periplasmic production considerably as compared to the wild-type. Our work, finally, demonstrates that periplasmic expression for SmbP-tagged proteins is not limited to the Sec pathway, in that the TorA-SmbP construct can export reasonable quantities of folded proteins to the periplasm. Although the Sec route has been the most widely used, sometimes, depending on the nature of the protein of interest, for example, if it contains cofactors, it is more appropriate to consider using the Tat route over the Sec. SmbP therefore can be recommended in terms of its particular versatility when combined with signal peptides for the two different routes.

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

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  1. Gopal, G. J., & Kumar, A. (2013). Strategies for the production of recombinant protein in Escherichia coli. The Protein Journal, 32(6), 419–425.

    Article  CAS  PubMed  Google Scholar 

  2. Terpe, K. (2006). Overview of bacterial expression systems for heterologous protein production: From molecular and biochemical fundamentals to commercial systems. Applied Microbiology and Biotechnology, 72(2), 211–222.

    Article  CAS  PubMed  Google Scholar 

  3. Vargas-Cortez, T., Morones-Ramirez, J. R., Balderas-Renteria, I., & Zarate, X. (2016). Expression and purification of recombinant proteins in Escherichia coli tagged with a small metal-binding protein from Nitrosomonas europaea. Protein Expression and Purification, 118, 49–54.

    Article  CAS  PubMed  Google Scholar 

  4. Barney, B. M., LoBrutto, R., & Francisco, W. A. (2004). Characterization of a small metal binding protein from Nitrosomonas europaea. Biochemistry, 43(35), 11206–11213.

    Article  CAS  PubMed  Google Scholar 

  5. Dow, B. A., Tatulian, S. A., & Davidson, V. L. (2015). Use of the amicyanin signal sequence for efficient periplasmic expression in E. coli of a human antibody light chain variable domain. Protein Expression and Purification, 108, 9–12.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. de Marco, A. (2015). Recombinant antibody production evolves into multiple options aimed at yielding reagents suitable for application-specific needs. Microbial Cell Factories, 14(1), 125.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Ramanan, R. N., Tan, J. S., Mohamed, M. S., Ling, T. C., Tey, B. T., & Ariff, A. B. (2010). Optimization of osmotic shock process variables for enhancement of the release of periplasmic interferon-a2b from Escherichia coli using response surface method. Process Biochemistry, 45(2), 196–202.

    Article  CAS  Google Scholar 

  8. Mori, H., & Ito, K. (2001). The Sec protein-translocation pathway. Trends in Microbiology, 9(10), 494–500.

    Article  CAS  PubMed  Google Scholar 

  9. Natale, P., Brüser, T., & Driessen, A. J. M. (2008). Sec- and Tat-mediated protein secretion across the bacterial cytoplasmic membrane-Distinct translocases and mechanisms. Biochimica et Biophysica Acta - Biomembranes, 1778(9), 1735–1756.

    Article  CAS  Google Scholar 

  10. Thomas, J. D., Daniel, R. A., Errington, J., & Robinson, C. (2001). Export of active green fluorescent protein to the periplasm by the twin-arginine translocase (Tat) pathway in Escherichia coli. Molecular Microbiology, 39(1), 47–53.

    Article  CAS  PubMed  Google Scholar 

  11. Patel, R., Smith, S. M., & Robinson, C. (2014). Protein transport by the bacterial Tat pathway. Biochimica et Biophysica Acta - Molecular Cell Research, 1843(8), 1620–1628.

    Article  CAS  Google Scholar 

  12. Cantu-Bustos, J. E., Vargas-Cortez, T., Morones-Ramirez, J. R., Balderas-Renteria, I., Galbraith, D. W., McEvoy, M. M., et al. (2016). Expression and purification of recombinant proteins in Escherichia coli tagged with the metal-binding protein CusF. Protein Expression and Purification, 121, 61–65.

    Article  CAS  PubMed  Google Scholar 

  13. Schneider, C. A., Rasband, W. S., & Eliceiri, K. W. (2012). NIH Image to ImageJ: 25 years of image analysis. Nature Methods, 9(7), 671–675.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Remmert, M., Lopez, R., Li, W., Dineen, D., Sievers, F., Gibson, T. J., et al. (2011). Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Molecular Systems Biology, 7(1), 539.

    Article  Google Scholar 

  15. Schierle, C. F., Berkmen, M., Huber, D., Kumamoto, C., Boyd, D., & Beckwith, J. (2003). The DsbA signal sequence directs efficient, cotranslational export of passenger proteins to the Escherichia coli periplasm via the signal recognition particle pathway. Journal of Bacteriology, 185(19), 5706–5713.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Cristóbal, S., De Gier, J. W., Nielsen, H., & Von Heijne, G. (1999). Competition between Sec- and Tat-dependent protein translocation in Escherichia coli. EMBO Journal, 18(11), 2982–2990.

    Article  PubMed  Google Scholar 

  17. Burgess, R. R. (2009). Preparing a Purification Summary Table. In Methods in Enzymology, Volume 463 Guide to Protein Purification, Second Edition (pp. 29-34). Academic Press.

  18. Cantu-Bustos, J. E., Cano del Villar, K. D., Vargas-Cortez, T., Morones-Ramirez, J. R., Balderas-Renteria, I., & Zarate, X. (2016). Recombinant protein production data after expression in the bacterium Escherichia coli. Data in Brief, 7, 502–508.

    Article  PubMed  PubMed Central  Google Scholar 

  19. Loftin, I. R., Franke, S., Roberts, S. A., Weichsel, A., Héroux, A., Montfort, W. R., et al. (2005). A novel copper-binding fold for the periplasmic copper resistance protein CusF. Biochemistry, 44(31), 10533–10540.

    Article  CAS  PubMed  Google Scholar 

  20. Astashkin, A. V., Raitsimring, A. M., Walker, F. A., Rensing, C., & McEvoy, M. M. (2005). Characterization of the copper(II) binding site in the pink copper binding protein CusF by electron paramagnetic resonance spectroscopy. Journal of biological inorganic chemistry: JBIC: a publication of the Society of Biological Inorganic Chemistry, 10(3), 221–230.

    Article  CAS  Google Scholar 

  21. Sockolosky, J. T., & Szoka, F. C. (2013). Periplasmic production via the pET expression system of soluble, bioactive human growth hormone. Protein Expression and Purification, 87(2), 129–135.

    Article  CAS  PubMed  Google Scholar 

  22. Dong, Z., Zhang, J., Du, G., Chen, J., Li, H., & Lee, B. (2015). Periplasmic export of bile salt hydrolase in Escherichia coli by the Twin-Arginine signal peptides. Applied Biochemistry and Biotechnology, 177(2), 458–471.

    Article  CAS  PubMed  Google Scholar 

  23. Zhou, Y., Zhou, Y., Li, J., Chen, J., Yao, Y., Yu, L., et al. (2015). Efficient expression, purification and characterization of native human cystatin C in Escherichia coli periplasm. Protein Expression and Purification, 111, 18–22.

    Article  CAS  PubMed  Google Scholar 

  24. Samant, S., Gupta, G., Karthikeyan, S., Haq, S. F., Nair, A., Sambasivam, G., & Sukumaran, S. (2014). Effect of codon-optimized E. coli signal peptides on recombinant Bacillus stearothermophilus maltogenic amylase periplasmic localization, yield and activity. Journal of Industrial Microbiology and Biotechnology, 41(9), 1435–1442.

  25. Takemori, D., Yoshino, K., Eba, C., Nakano, H., & Iwasaki, Y. (2012). Extracellular production of phospholipase A 2 from Streptomyces violaceoruber by recombinant Escherichia coli. Protein Expression and Purification, 81(2), 145–150.

    Article  CAS  PubMed  Google Scholar 

  26. Mohajeri, A., Pilehvar-Soltanahmadi, Y., Pourhassan-Moghaddam, M., Abdolalizadeh, J., Karimi, P., & Zarghami, N. (2016). Cloning and expression of recombinant human endostatin in periplasm of Escherichia coli expression system. Advanced Pharmaceutical Bulletin, 6(2), 187–194.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Freudl, R. (2017). Beyond amino acids: Use of the Corynebacterium glutamicum cell factory for the secretion of heterologous proteins. Journal of Biotechnology, 258, 101–109.

    Article  CAS  PubMed  Google Scholar 

  28. Santini, C. L., Bernadac, A., Zhang, M., Chanal, A., Ize, B., Blanco, C., et al. (2001). Translocation of jellyfish Green Fluorescent Protein via the Tat system of Escherichia coli and change of its periplasmic localization in response to osmotic up-shock. Journal of Biological Chemistry, 276(11), 8159–8164.

    Article  CAS  PubMed  Google Scholar 

  29. Dammeyer, T., & Tinnefeld, P. (2012). Engineered fluorescent proteins illuminate the bacterial periplasm. Computational and Structural Biotechnology Journal, 3(4), e201210013.

    Article  PubMed  PubMed Central  Google Scholar 

  30. He, X. M., Yuan, B. F., & Feng, Y. Q. (2017). Facial synthesis of nickel(II)-immobilized carboxyl cotton chelator for purification of histidine-tagged proteins. Journal of Chromatography, B: Analytical Technologies in the Biomedical and Life Sciences, 1043, 122–127.

    Article  CAS  PubMed  Google Scholar 

  31. Zárate, X., Henderson, D. C., Phillips, K. C., Lake, A. D., & Galbraith, D. W. (2010). Development of high-yield autofluorescent protein microarrays using hybrid cell-free expression with combined Escherichia coli S30 and wheat germ extracts. Proteome Science, 8(1), 32.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Low, K. O., Mahadi, N. M., & Illias, R. M. (2013). Optimisation of signal peptide for recombinant protein secretion in bacterial hosts. Applied Microbiology and Biotechnology, 97(9), 3811–3826.

    Article  CAS  PubMed  Google Scholar 

  33. Sletta, H., Tøndervik, A., Hakvåg, S., Vee Aune, T. E., Nedal, A., Aune, R., et al. (2007). The presence of N-terminal secretion signal sequences leads to strong stimulation of the total expression levels of three tested medically important proteins during high-cell-density cultivations of Escherichia coli. Applied and Environmental Microbiology, 73(3), 906–912.

    Article  CAS  PubMed  Google Scholar 

  34. Suominen, I., Meyer, P., Tilgmann, C., Glumoff, T., Glumoff, V., Kapyla, J., et al. (1995). Effects of signal peptide mutations on processing of Bacillus stearothermophilus a-amylase in Escherichia coli. Microbiology, 141(3), 649–654.

    Article  CAS  PubMed  Google Scholar 

  35. Adams, H., Scotti, P. A., De Cock, H., Luirink, J., & Tommassen, J. (2002). The presence of a helix breaker in the hydrophobic core of signal sequences of secretory proteins prevents recognition by the signal-recognition particle in Escherichia coli. European Journal of Biochemistry, 269(22), 5564–5571.

    Article  CAS  PubMed  Google Scholar 

  36. Green, E. R., & Mecsas, J. (2016). Bacterial Secretion Systems: An Overview. In Virulence Mechanisms of Bacterial Pathogens, Fifth Edition (pp. 215–239). American Society of Microbiology.

  37. Heim, R., & Tsien, R. Y. (1996). Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer. Current Biology, 6(2), 178–182.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank Mexico’s Consejo Nacional de Ciencia y Tecnologia (CONACYT) for the financial support provided to BDS during his graduate studies. This work was funded by Grants UANL-PAICYT-2015 CN567-15 and CONACYT Grant CB 2012-179,774-B awarded to XZ.

Author information

Authors and Affiliations

  1. Universidad Autonoma de Nuevo Leon, Facultad de Ciencias Quimicas, 66455, San Nicolas de los Garza, NL, Mexico

    Bryan D. Santos, Jose Ruben Morones-Ramirez, Isaias Balderas-Renteria & Xristo Zarate

  2. Universidad Autonoma de Nuevo Leon, Centro de Investigacion en Biotecnologia y Nanotecnologia, Facultad de Ciencias Quimicas, Parque de Investigacion e Innovacion Tecnologica, 66629, Apodaca, NL, Mexico

    Jose Ruben Morones-Ramirez, Isaias Balderas-Renteria & Xristo Zarate

  3. Departamento de Patologia Clinica, Universidad Autonoma de Nuevo Leon, Hospital Universitario Dr. Jose Eleuterio Gonzalez, 64460, Monterrey, NL, Mexico

    Nestor G. Casillas-Vega

  4. School of Plant Sciences and BIO5 Institute, University of Arizona, Tucson, AZ, 85721, USA

    David W. Galbraith

Authors
  1. Bryan D. Santos
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jose Ruben Morones-Ramirez
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Isaias Balderas-Renteria
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Nestor G. Casillas-Vega
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. David W. Galbraith
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Xristo Zarate
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Xristo Zarate.

Ethics declarations

Conflict of interest

The authors declare no conflict of interest.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Santos, B.D., Morones-Ramirez, J.R., Balderas-Renteria, I. et al. Optimizing Periplasmic Expression in Escherichia coli for the Production of Recombinant Proteins Tagged with the Small Metal-Binding Protein SmbP. Mol Biotechnol 61, 451–460 (2019). https://doi.org/10.1007/s12033-019-00176-4

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12033-019-00176-4

Keywords

Search

Navigation