A Generic Protocol for Intracellular Expression of Recombinant Proteins in Bacillus subtilis

  • Trang Phan
  • Phuong Huynh
  • Tuom Truong
  • Hoang Nguyen
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1586)

Abstract

Bacillus subtilis (B. subtilis) is a potential and attractive host for the production of recombinant proteins. Different expression systems for B. subtilis have been developed recently, and various target proteins have been recombinantly synthesized and purified using this host. In this chapter, we introduce a generic protocol to express a recombinant protein in B. subtilis. It includes protocols for (1) using our typical expression vector (plasmid pHT254) to introduce a target gene, (2) transformation of the target vector into B. subtilis, and (3) evaluation of the actual expression of a recombinant protein.

Key words

Bacillus subtilis pHT01 vector Pgrac promoter pHT254 Pgrac100 promoter 

Notes

Acknowledgments

This research was funded by the Vietnam National Foundation for Science and Technology Development (Nafosted) under grant number 106-NN.02-2015.24. The research was also supported by TWAS under research grants 14-201 RG/BIO/AS_G.

References

  1. 1.
    Adrio JL, Demain AL (2010) Recombinant organisms for production of industrial products. Bioeng Bugs 1:116–131CrossRefPubMedGoogle Scholar
  2. 2.
    Demain AL, Vaishnav P (2009) Production of recombinant proteins by microbes and higher organisms. Biotechnol Adv 27:297–306CrossRefPubMedGoogle Scholar
  3. 3.
    Sahdev S, Khattar SK, Saini KS (2008) Production of active eukaryotic proteins through bacterial expression systems: a review of the existing biotechnology strategies. Mol Cell Biochem 307:249–264CrossRefPubMedGoogle Scholar
  4. 4.
    Schallmey M, Singh A, Ward OP (2004) Developments in the use of Bacillus species for industrial production. Can J Microbiol 50:1–17CrossRefPubMedGoogle Scholar
  5. 5.
    Schumann W (2007) Production of recombinant proteins in Bacillus subtilis. Adv Appl Microbiol 62:137–189CrossRefPubMedGoogle Scholar
  6. 6.
    Tjalsma H, Antelmann H, Jongbloed JDH et al (2004) Proteomics of protein secretion by Bacillus subtilis: separating the “secrets” of the secretome. Microbiol Mol Biol Rev 68:207–233CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Nguyen HD, Nguyen QA, Ferreira RC et al (2005) Construction of plasmid-based expression vectors for Bacillus subtilis exhibiting full structural stability. Plasmid 54:241–248CrossRefPubMedGoogle Scholar
  8. 8.
    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 46:189–195CrossRefPubMedGoogle Scholar
  9. 9.
    Nguyen HD, Phan TTP, Schumann W (2007) Expression vectors for the rapid purification of recombinant proteins in Bacillus subtilis. Curr Microbiol 55:89–93CrossRefPubMedGoogle Scholar
  10. 10.
    Phan TTP, Nguyen HD, Schumann W (2012) Development of a strong intracellular expression system for Bacillus subtilis by optimizing promoter elements. J Biotechnol 157:167–172CrossRefPubMedGoogle Scholar
  11. 11.
    Phan TTP, Nguyen HD, Schumann W (2013) Construction of a 5′-controllable stabilizing element (CoSE) for over-production of heterologous proteins at high levels in Bacillus subtilis. J Biotechnol 168:32–39CrossRefPubMedGoogle Scholar
  12. 12.
    Phan TTP, Tran LT, Schumann W et al (2015) Development of Pgrac100-based expression vectors allowing high protein production levels in Bacillus subtilis and relatively low basal expression in Escherichia coli. Microb Cell Fact 14:72CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Hirata H, Fukazawa T, Negoro S et al (1986) Structure of a beta-galactosidase gene of Bacillus stearothermophilus. J Bacteriol 166:722–727CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    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 170:117–122CrossRefPubMedGoogle Scholar
  15. 15.
    Inoue H, Nojima H, Okayama H (1990) High efficiency transformation of Escherichia coli with plasmids. Gene 96:23–28CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Trang Phan
    • 1
  • Phuong Huynh
    • 1
  • Tuom Truong
    • 1
  • Hoang Nguyen
    • 1
  1. 1.VNUHCM-University of ScienceHochiminh CityVietnam

Personalised recommendations