Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

Extracellular secretion of polypeptides using a modified Escherichia coli flagellar secretion apparatus

Nature Biotechnology volume 23, pages 475–481 (2005)Cite this article

Abstract

We developed a modified flagellar type III secretion apparatus to secrete heterologous polypeptides into the growth medium of Escherichia coli. The secretion was facilitated by fusing the 173-bp untranslated DNA fragment upstream of the gene fliC (encoding flagellin) as well as a transcriptional terminator from fliC, into the gene encoding the polypeptide of interest. The polypeptides secreted into the growth medium at concentrations ranging from 1 to 15 mg/l were from Campylobacter jejuni (262 residues in length), Streptococcus pneumoniae (434 residues), Staphylococcus aureus (115 residues), and N-terminal FliC hybrid proteins, for example, the eukaryotic green fluorescent protein (238 residues). The expressed proteins represented >50% of total secreted protein. Previously reported protein yields from extracellular secretion of foreign proteins in E. coli have been low, approximately 100 μg/l1. The strengths of our method are the concentration and purity of the secreted proteins and its versatility with regard to the proteins' length and origin.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Schematic presentation of the plasmids, the gene constructs expressed in E. coli MKS12 and the secretion of the resulting polypeptides into culture medium.
Figure 2: Analysis of intracellular and secreted polypeptides from recombinant strains of E. coli: MKS12 expressing FliC fusion polypeptides and analysis of binding properties of the fusion polypeptides.
Figure 3: Analysis of intracellular and secreted polypeptides from recombinant strains of E. coli MKS12 expressing foreign polypeptides.

Similar content being viewed by others

Accession codes

Accessions

GenBank/EMBL/DDBJ

References

  1. Choi, J.H. & Lee, S.Y. Secretory and extracellular production of recombinant proteins using Escherichia coli. Appl. Microbiol. Biotechnol. 64, 625–635 (2004).

    Article  CAS  Google Scholar 

  2. Thanassi, D.G. & Hultgren, S.J. Multiple pathways allow secretion across the bacterial outer membrane. Curr. Opin. Cell Biol. 12, 420–430 (2000).

    Article  CAS  Google Scholar 

  3. Thomas, N.A. & Finley, B.B. Establishing order for type III secretion substrates—a hierarchial process. Trends Microbiol. 11, 398–403.

  4. Stebbins, C.E. & Galán, J. Maintenance of an unfolded polypeptide by a cognate chaperone in bacterial type III secretion. Nature 414, 77–81 (2001).

    Article  CAS  Google Scholar 

  5. Blocker, A., Komoriya, K. & Aizawa, S.-I. Type III secretion systems and bacterial flagella: insights into their function from structural similarities. Proc. Natl. Acad. Sci. USA 100, 3021–3030 (2003).

    Article  Google Scholar 

  6. Minamino, T. & Namba, K. Self-assembly and type III protein export of the bacterial flagellum. J. Mol. Microbiol. Biotechnol. 7, 5–17 (2004).

    Article  CAS  Google Scholar 

  7. Anderson, D.M. & Schneewind, O. Yersinia enterocolitica type III secretion: an mRNA signal that couples translation and secretion of YopQ. Mol. Microbiol. 31, 1139–1148 (1999).

    Article  CAS  Google Scholar 

  8. Lloyd, S.A., Norman, M., Rosqvist, R. & Wolf-Watz, H. Yersinia YopE is targeted for type III secretion by N-terminal, not mRNA, signals. Mol. Microbiol. 39, 520–531 (2001).

    Article  CAS  Google Scholar 

  9. Aldridge, P. & Hughes, K.T. Regulation of flagellar assembly. Curr. Opin. Microbiol. 5, 160–165 (2002).

    Article  CAS  Google Scholar 

  10. Parsot, C., Hamiaux, C. & Page, A.-L. The various and varying roles of specific chaperones in type III secretion systems. Curr. Opin. Microbiol. 6, 7–14 (2003).

    Article  CAS  Google Scholar 

  11. Evdokimov, A.G. et al. Similar modes of polypeptide recognition by export chaperones in flagellar biosynthesis and type III secretion. Nat. Struct. Biol. 10, 789–793 (2003).

    Article  CAS  Google Scholar 

  12. Young, G.M., Schmiel, D.H. & Miller, V.L. A new pathway for the secretion of virulence factors by bacteria: The flagellar export apparatus functions as a protein-secretion system. Proc. Natl. Acad. Sci. USA 96, 6456–6461 (1999).

    Article  CAS  Google Scholar 

  13. Lee, S.H. & Galán, J.A. Salmonella type III secretion-associated chaperones confer secretion-pathway specificity. Mol. Microbiol. 51, 483–495 (2004).

    Article  CAS  Google Scholar 

  14. Konkel, M.E. et al. Secretion of virulence proteins from Campylobacter jejuni is dependent on a functional flagellar export apparatus. J. Bacteriol. 186, 3296–3303 (2004).

    Article  CAS  Google Scholar 

  15. Chilcott, G.S. & Hughes, K.T. Coupling of flagellar gene expression to flagellar assembly in Salmonella enterica serovar Typhiurium and Escherichia coli. Microbiol. Mol. Biol. Rev. 64, 694–708 (2000).

    Article  CAS  Google Scholar 

  16. Karlinsey, J.E. et al. Completion of the hook-basal body complex of the Salmonella typhimurium flagellum is coupled to FlgM secretion and fliC transcription. Mol. Microbiol. 37, 1220–1231 (2000).

    Article  CAS  Google Scholar 

  17. Yonekura, K., Maki-Yonekura, S. & Namba, K. Complete atomic model of the bacterial flagellar filament by electron cryomicroscopy. Nature 424, 643–650 (2003).

    Article  CAS  Google Scholar 

  18. Blomfield, I.C., McClain, M.S. & Eisenstein, B.I. Type 1 fimbriae mutants of Escherichia coli K12: characterization of recognized afimbriate strains and construction of new fim deletion mutants. Mol. Microbiol. 5, 1439–1445 (1991).

    Article  CAS  Google Scholar 

  19. Westerlund-Wikström, B. et al. Functional expression of adhesive peptides as fusions to Escherichia coli flagellin. Prot. Engin. 10, 1319–1326 (1997).

    Article  Google Scholar 

  20. Signäs, C. et al. Nucleotide sequence of the gene for fibronectin-binding protein from Staphylococcus aureus: use of this peptide sequence in the synthesis of biologically active peptides. Proc. Natl. Acad. Sci. USA 86, 699–703 (1989).

    Article  Google Scholar 

  21. Blattner, F.R. et al. The complete genome sequence of Escherichia coli K12. Science 277, 1453–1463 (1997).

    Article  CAS  Google Scholar 

  22. Kuwajima, G. et al. Export of an N-terminal fragment of Escherichia coli flagellin by a flagellum-specific pathway. Proc. Natl. Acad. Sci. USA 86, 4953–4957 (1989).

    Article  CAS  Google Scholar 

  23. Ide, N., Ikebe, T. & Kutsukake, K. Reevaluation of the promoter structure of the class 3 flagellar operons of Escherichia coli and Salmonella. Genes Genet. Syst. 74, 113–116 (1999).

    Article  CAS  Google Scholar 

  24. Pei, Z. & Blaser, M.J. PEB1, the major cell-binding factor of Campylobacter jejuni, is a homolog of the binding component in Gram-negative nutrient transport systems. J. Biol. Chem. 268, 18717–18725 (1993).

    CAS  PubMed  Google Scholar 

  25. Bergmann, S. et al. Identification of a novel plasmin(ogen)-binding motif in surface displayed α-enolase of Streptococcus pneumoniae. Mol. Microbiol. 49, 411–423 (2003).

    Article  CAS  Google Scholar 

  26. Davis, S. & Vierstra, R.D. Soluble, highly fluorescent variants of green fluorescent protein (GFP) for use in higher plants. Plant Mol. Biol. 36, 521–528 (1998).

    Article  CAS  Google Scholar 

  27. Hopp, T.V. et al. A short polypeptide marker sequence useful for recombinant protein identification and purification. BioTechnology 6, 1204–1210 (1988).

    Article  CAS  Google Scholar 

  28. Deckers, D. et al. Periplasmic lysozyme inhibitor contributes to lysozyme resistance in Escherichia coli. Cell. Mol. Life Sci. 61, 1229–1237 (2004).

    Article  CAS  Google Scholar 

  29. Nikaido, H. Outer membrane barrier as a mechanism of antimicrobial resistance. Antimicrob. Agents Chemother. 33, 1831–1836 (1989).

    Article  CAS  Google Scholar 

  30. Hirano, T., Minamino, T., Namba, K. & Macnab, R.M. Substrate specificity classes and the recognition for Salmonella type III flagellar export. J. Bacteriol. 185, 2485–2492 (2003).

    Article  CAS  Google Scholar 

  31. Sambrook, J. & Russell, D. Molecular Cloning: A Laboratory Manual 3rd edn. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA, 2001).

    Google Scholar 

  32. Donnenberg, M. & Kaper, J.B. Construction of an eae deletion mutant of enteropathogenic Escherichia coli by using a positive-selection suicide vector. Infect. Immun. 59, 4310–4317 (1991).

    Article  CAS  Google Scholar 

  33. Mobley, H.L.T. et al. Isogenic P-fimbrial deletion mutants of pyelonephritogenic Escherichia coli: the role of αGal(1-4)βGal binding virulence of a wild-type strain. Mol. Microbiol. 10, 143–155 (1993).

    Article  CAS  Google Scholar 

  34. Kovach, M.E. et al. pBBR1MCS: a broad-host-range cloning vector. Biotechniques. 16, 800–802 (1994).

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank I. Blomfield for the E. coli strain MG1655 Δfim; H. Rautelin for the C. jejuni strain T-71431; H. Saarilahti for the plasmid psmGfp; and R. Lameranta, K. Alsti, L. Partanen and T. Vesa for technical assistance. This work was supported by the National Technology Agency (Tekes, grant number 40312/03), University of Helsinki (grant number 2105045) and the Academy of Finland (grant numbers 78141 and 202009, and the Microbes and Man Program).

Author information

Authors and Affiliations

  1. General Microbiology, Faculty of Biosciences, P.O. Box 56, FIN-00014 University of Helsinki, Helsinki, Finland

    Katariina Majander, Lena Anton, Jenni Antikainen, Hannu Lång, Timo K Korhonen & Benita Westerlund-Wikström

  2. Orion Diagnostica Oy, P.O. Box 83, Espoo, FIN-02101, Finland

    Mirko Brummer

Authors
  1. Katariina Majander
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Lena Anton
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jenni Antikainen
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Hannu Lång
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Mirko Brummer
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Timo K Korhonen
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Benita Westerlund-Wikström
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Benita Westerlund-Wikström.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Majander, K., Anton, L., Antikainen, J. et al. Extracellular secretion of polypeptides using a modified Escherichia coli flagellar secretion apparatus. Nat Biotechnol 23, 475–481 (2005). https://doi.org/10.1038/nbt1077

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt1077

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing