Current Microbiology

, Volume 55, Issue 2, pp 89–93 | Cite as

Expression Vectors for the Rapid Purification of Recombinant Proteins in Bacillus subtilis

  • Hoang Duc Nguyen
  • Trang Thi Phuong Phan
  • Wolfgang SchumannEmail author


We describe the construction of six novel plasmid-based IPTG-inducible expression vectors for Bacillus subtilis and related species. While one vector allows intracellular production of recombinant proteins, the second provides a strong secretion signal. The third vector allows addition of the c-Myc epitope tag, and the remaining three vectors provide the purification tags His and Strep. The versatility of all six vectors was demonstrated by the insertion of several reporter genes and by their regulated overexpression. Recombinant proteins with a His- or Strep-tag could be purified to near homogeneity in a single step.


Recombinant Protein Listeria Monocytogenes Direct Repeat Structural Instability Small Plasmid 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



This work was supported by the DLR (VNB02/B03), the MOST (Life Science-643204), and the Bayerische Forschungsstiftung grants to T.T.P.P. All vectors can be ordered from Mobitec (

Literature Cited

  1. 1.
    Anderson RP, Roth JR (1977) Tandem genetic duplications in phage and bacteria. Annu Rev Microbiol 31:473–505PubMedCrossRefGoogle Scholar
  2. 2.
    Cornet P, Millet J, Béguin P, Aubert JP (1983) Characterization of two cel (cellulose degradation) genes of Clostridium thermocellum coding for endogluanases. Bio/Technology 1:589–594CrossRefGoogle Scholar
  3. 3.
    Evan GI, Lewis GK, Ramsay G, Bishop JM (1985) Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product. Mol Cell Biol 5:3610–3616PubMedGoogle Scholar
  4. 4.
    Harwood CR (1992) Bacillus subtilis and its relatives: molecular biological and industrial workhorses. Trends Biotechnol 10:247–256PubMedCrossRefGoogle Scholar
  5. 5.
    Hirata H, Fukazawa T, Negoro S, Okada H (1986) Structure of a β-galactosidase gene of Bacillus stearothermophilus. J Bacteriol 166:722–727PubMedGoogle Scholar
  6. 6.
    Hochuli E (1990) Purification of recombinant proteins with metal chelate adsorbent. Genet Eng (NY) 12:87–98Google Scholar
  7. 7.
    Homuth G, Masuda S, Mogk A, Kobayashi Y, Schumann W (1997) The dnaK operon of Bacillus subtilis is heptacistronic. J Bacteriol. 179:1153–1164PubMedGoogle Scholar
  8. 8.
    Junttila MR, Saarinen S, Schmidt T, Kast J, Westermarck J (2005) Single-step Strep-tag purification for the isolation and identification of protein complexes from mammalian cells. Proteomics 5:1199–1203PubMedCrossRefGoogle Scholar
  9. 9.
    Kaltwasser M, Wiegert T, Schumann W (2001) Construction and application of epitope- and GFT-tagging integration vectors for Bacillus subtilis. Appl Environ Microbiol 68:2624–2628CrossRefGoogle Scholar
  10. 10.
    Kim L, Mogk A, Schumann W (1996) A xylose-inducible Bacillus subtilis integration vector and its application. Gene 181:71–76PubMedCrossRefGoogle Scholar
  11. 11.
    Meima R, Van Dijl JM, Bron S (2004) Expression systems in Bacillus. In F Baneyx (ed) Protein expression technologies. Horizon Bioscience, Norfold, UK pp 199–252Google Scholar
  12. 12.
    Nguyen DH, Nguyen QA, Ferreira RC, Ferreira LCS, Tran LT, Schumann W. (2004) Construction of plasmid-based expression vectors for Bacillus subtilis. Plasmid 54:241–248CrossRefGoogle Scholar
  13. 13.
    Nguyen HD, Schumann W (2006) Establishment of an experimental system allowing immobilization of proteins on the surface of Bacillus subtilis cells. J Biotechnol 122:473–482PubMedCrossRefGoogle Scholar
  14. 14.
    Pallen MJ, Lam AC, Antonio M, Dunbar K (2001) An embarrassment of sortases: a richness of substrates? Trends Microbiol 9:97–101PubMedCrossRefGoogle Scholar
  15. 15.
    Palva I (1982) Molecular cloning of alpha-amylase gene from Bacillus amyloliquefaciens and its expression in B. subtilis. Gene 19:81–87PubMedCrossRefGoogle Scholar
  16. 16.
    Phan TTP, Nguyen HD, Schumann W (2006) Novel plasmid-based expression vectors for intra- and extracellular production of recombinant proteins in Bacillus subtilis. Protein Expr Purif 46:189–195PubMedCrossRefGoogle Scholar
  17. 17.
    Ripio MT, Dominguez-Bernal G, Suarez M, Brehem K, Berche P, Vazquez-Boland JA (1996) Transcriptional activation of virulence genes in wild-type strains of Listeria monocytogenes in response to a change in the extracellular medium composition. Res Microbiol 147:371–378PubMedCrossRefGoogle Scholar
  18. 18.
    Saito H, Shibata T, Ando T (1979) Mapping of genes determining nonpermissiveness and host-specific restriction to bacteriophages in Bacillus subtilis Marburg. Mol Gen Genet 170:117–122PubMedCrossRefGoogle Scholar
  19. 19.
    Schallmey M, Singh A, Ward OP (2004) Developments in the use of Bacillus species for industrial production. Can J Microbiol 50:1–17PubMedCrossRefGoogle Scholar
  20. 20.
    Schmidt TG, Koepke J, Frank R, Skerra A (1996) Molecular interaction between the Strep-tag affinity peptide and its cognate target, streptavidin. J Mol Biol 255:753–766PubMedCrossRefGoogle Scholar
  21. 21.
    Schmidt TG, Skerra A (1993) The random peptide library-assisted engineering of a C-terminal affinity peptide, useful for the detection and purification of a functional Ig Fv fragment. Protein Eng 6:109–122PubMedCrossRefGoogle Scholar
  22. 22.
    Scholz Q, Thiel A, Hillen W, Niederweis M (2000) Quantitative analysis of gene expression with an improved green fluorescent protein. Eur J Biochem 267:1565–1570PubMedCrossRefGoogle Scholar
  23. 23.
    Schulz A, Schwab S, Versteeg S, Schumann W (1997) The htpG gene of Bacillus subtilis belongs to class III heat shock genes and is under negative control. J Bacteriol 10:3103–3109Google Scholar
  24. 24.
    Titok MA, Chapuis J, Selezneva YV, Lagodich AV, Prokulevich VA, Ehrlich SD, Jannière L (2003) Bacillus subtilis soil isolates: plasmid replicon analysis and construction of a new theta-replicating vector. Plasmid 49:53–62PubMedCrossRefGoogle Scholar
  25. 25.
    Westers L, Westers H, Quax WJ (2004) Bacillus subtilis as cell factory for pharmaceutical proteins: a biotechnological approach to optimize the host organism. Biochim Biophys Acta 1694:299–310PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2007

Authors and Affiliations

  • Hoang Duc Nguyen
    • 1
  • Trang Thi Phuong Phan
    • 1
  • Wolfgang Schumann
    • 1
    Email author
  1. 1.Institute of GeneticsUniversity of BayreuthBayreuthGermany

Personalised recommendations