Skip to main content
SpringerLink
Log in

Construction of an Expression Vector for Production and Purification of Human Somatostatin in Escherichia coli

  • Research
  • Published:
Molecular Biotechnology Aims and scope Submit manuscript

Abstract

Somatostatin/growth hormone-inhibiting hormone is the peptide that inhibits secretion of somatotropin/growth hormone. Solid-phase synthesis methods are being currently used to produce somatostatin. Recombinant peptide synthesis is widely described for the production of small proteins and peptides; however, the production at industrial scale of peptides for biopharmaceutical applications is limited for economic reasons. Here, we propose the use of a new pGB-SMT plasmid to produce Somatostatin, as a C-terminal fusion protein with a Kluyveromyces lactis β-galactosidase fragment. To facilitate removal of that fragment by CNBr cleavage, a methionine residue was introduced at the N-terminal of the hormone peptide. The use of this construction enables an IPTG-free expression system. The suitability of this procedure has been assessed in a 15 l scale-up experiment yielding almost 300 mg, with purity >99 % and it is being implemented for commercial scale. The plasmid pGB-SMT here described is an alternative option for a cheap and high expression of other short peptide hormones.

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
Fig. 6
Fig. 7

Similar content being viewed by others

References

  1. Patel, Y. C. (1999). Somatostatin and its receptor family. Frontiers in Neuroendocrinology, 20, 157–198.

    Article  CAS  Google Scholar 

  2. Zeng, Y. X., Du, N. X., & Tan, J. Y. (1991). Expression of somatostatin gene in Escherichia coli D29A1. Yi Chuan Xue Bao, 18, 282–288.

    CAS  Google Scholar 

  3. Patel, Y. C., & Reichlin, S. (1978). Somatostatin in hypothalamus, extrahypothalamic brain, and peripheral tissues of the rat. Endocrinology, 102, 523–530.

    Article  CAS  Google Scholar 

  4. Mentlein, R., Eichler, O., Forstreuter, F., & Held-Feindt, J. (2001). Somatostatin inhibits the production of vascular endothelial growth factor in human glioma cells. International Journal of Cancer, 92, 545–550.

    Article  CAS  Google Scholar 

  5. Reynaert, H., & Geerts, A. (2003). Pharmacological rationale for the use of somatostatin and analogues in portal hypertension. Aliment Pharmacology and Therapeutics, 18, 375–386.

    Article  CAS  Google Scholar 

  6. Albericio, F. (2004). Developments in peptide and amide synthesis. Current Opinion in Chemical Biology, 8, 211–221.

    Article  CAS  Google Scholar 

  7. Bruckdorfer, T., Marder, O., & Albericio, F. (2004). From production of peptides in milligram amounts for research to multi-tons quantities for drugs of the future. Current Pharmaceutical Biotechnology, 5, 29–43.

    Article  CAS  Google Scholar 

  8. Loffet, A. (2002). Peptides as drugs, is there a market? Journal of Peptide Science, 8, 1–7.

    Article  CAS  Google Scholar 

  9. Itakura, K., Hirose, T., Crea, R., Riggs, A. D., Heyneker, H. L., Bolivar, F., et al. (1977). Expression in Escherichia coli of a chemically synthesized gene for the hormone somatostatin. Science, 198, 1056–1063.

    Article  CAS  Google Scholar 

  10. Karpova, S. K., Sazina, E. T., Karasev, V. S., Bader, L. B., Sergienko, O. V., Shishkina, A. A., et al. (1993). Synthesis of somatostatin in Escherichia coli cells, isolation and characteristics. Bioorganicheskaia Khimiia, 19, 612–622.

    CAS  Google Scholar 

  11. Hanahan, D., Jessee, J., & Bloom, F. R. (1991). Plasmid transformation of Escherichia coli and other bacteria. Methods in Enzymology, 204, 63–113.

    Article  CAS  Google Scholar 

  12. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680–685.

    Article  CAS  Google Scholar 

  13. Schägger, H., & von Jagow, G. (1987). Tricine-sodium dodecyl sulfate polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDalton. Analytical Biochemistry, 166, 368–379.

    Article  Google Scholar 

  14. Sambrook, J., & Russell, D. (2001). Molecular cloning, a laboratory manual (3rd ed.). Cold Spring Harbor: Cold Spring Harbor Laboratory Press.

    Google Scholar 

  15. Grosjean, H., & Fiers, W. (1982). Preferential codon usage in prokaryotic genes, the optimal codon-anticodon interaction energy and the selective codon usage in efficiently expressed genes. Gene, 18, 99–209.

    Article  Google Scholar 

  16. Rubio-Texeira, M., Castrillo, J. I., Adam, A. C., Ugalde, U. O., & Polaina, J. (1998). Highly efficient assimilation of lactose by a metabolically engineered strain of Saccharomyces cerevisiae. Yeast, 14, 827–837.

    Article  CAS  Google Scholar 

  17. Gros, E. (1967). The cyanogen bromide reaction. Methods in Enzymology, 11, 238–255.

    Article  Google Scholar 

  18. Juárez, P., Sanz, L., & Calvete, J. J. (2004). Snake venomics, characterization of protein families in Sistrurus barbouri venom by cysteine mapping, N-terminal sequencing, and tandem mass spectrometry analysis. Proteomics, 4, 327–338.

    Article  Google Scholar 

  19. Seed, B. (1987). An LFA-3 cDNA encodes a phospholipid-linked membrane protein homologous to its receptor CD2. Nature, 329, 840–842.

    Article  CAS  Google Scholar 

  20. Cserjan-Puschmann, M., Grabherr, R., Striedner, G., Clementschitsch, F., & Bayer, K. (2002). Optimizing recombinant microbial fermentation processes. Biopharma International, 15, 26–34.

    CAS  Google Scholar 

  21. Cserjan-Puschmann, M., Kramer, W., Duerrschmid, E., Striedner, G., & Bayer, K. (1999). Metabolic approaches for the optimisation of recombinant fermentation processes. Applied Microbiology and Biotechnology, 53, 43–50.

    Article  CAS  Google Scholar 

  22. Tolia, N. H., & Joshua-Tor, L. (2006). Strategies for protein coexpression in Escherichia coli. Nature Methods, 3, 55–64.

    Article  CAS  Google Scholar 

  23. Sadeghi, H. M., Rabbani, M., Rismani, E., Moazen, F., Khodabakhsh, F., Dormiani, K., et al. (2011). Optimization of the expression of reteplase in Escherichia coli. Res Pharm Sci, 6, 87–92.

    CAS  Google Scholar 

  24. Grindley, J., & Odgen, J. (2000). Understanding biopharmaceuticals, manufacturing and regulatory issues. Florida: CRC Press.

    Google Scholar 

  25. Hansen, L. H., Knudsen, S., & Sørensen, S. J. (1998). The effect of the lacY gene on the induction of IPTG inducible promoters, studied in Escherichia coli and Pseudomonas fluorescens. Current Microbiology, 36, 341–347.

    Article  CAS  Google Scholar 

  26. Studier, F. W. (2005). Protein production by auto-induction in high density shaking cultures. Protein Expression and Purification, 41, 207–234.

    Article  CAS  Google Scholar 

  27. Jensen, P. R., Westerhoff, H. V., & Michelsen, O. (1993). The use of lac-type promoters in control analysis. European Journal of Biochemistry, 211, 181–191.

    Article  CAS  Google Scholar 

  28. Choi, J. H., & Lee, S. Y. (2004). Secretory and extracellular production of recombinant proteins using Escherichia coli. Applied Microbiology and Biotechnology, 64, 625–635.

    Article  CAS  Google Scholar 

  29. Yoon, S. H., Kim, S. K., & Kim, J. F. (2010). Secretory production of recombinant proteins in Escherichia coli. Recent Patents on Biotechnology, 4, 23–29.

    Article  CAS  Google Scholar 

  30. Sauter, S. R., Diekmann, S., & Braun, V. (2003). Two-step purification of the outer membrane transporter and activator protein ShlB from Escherichia coli using internally His6-tagged constructs. Journal of Chromatography B Analytical Technologies Biomedical and Life Science, 25, 33–37.

    Article  Google Scholar 

  31. Dong, X. Y., Feng, X. D., & Sun, Y. (2010). His-tagged protein purification by metal-chelate affinity extraction with nickel-chelate reverse micelles. Biotechnology Progress, 26, 1088–1094.

    CAS  Google Scholar 

Download references

Acknowledgments

Authors are grateful to Dr. J. Polaina for kindly providing plasmid pMR11 and Dr J. J. Calvete for mass spectrometry analysis. We thank P. Vicente, Dr. F. Bosch and Emeritus Professor R. Sentandreu for stimulating support to this project. This project was supported by Génesis Especialidades Farmacéuticas y Biotecnología S.A. Fund, Spanish Government Fund (CDTI-05-0469) and Generalitat Valenciana Fund (IMPIVA-INCOMA-05-19). S. M. and A. N. were supported by Torres Quevedo Grants from the Ministerio de Ciencia e Innovación-Spanish Government.

Author information

Authors and Affiliations

  1. Departament de Microbiologia i Ecologia, Universitat de València, Dr. Moliner, 50, 46100, Burjassot, Spain

    Sergi Maicas, Almudena Nieto & Eulogio Valentín

  2. Laboratorio de Genética Molecular, Centro de Investigación “Príncipe Felipe”, Camino de las Moreras s/n, 46013, Valencia, Spain

    Ismaïl Moukadiri

Authors
  1. Sergi Maicas
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ismaïl Moukadiri
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Almudena Nieto
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Eulogio Valentín
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Sergi Maicas.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Maicas, S., Moukadiri, I., Nieto, A. et al. Construction of an Expression Vector for Production and Purification of Human Somatostatin in Escherichia coli . Mol Biotechnol 55, 150–158 (2013). https://doi.org/10.1007/s12033-013-9667-3

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12033-013-9667-3

Keywords

Search

Navigation