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Bacillus subtilis Expression System

Bacillus subtilis Expression System

Gram-positive bacteria are well known for their contributions to agricultural, medical and food biotechnology and for the production of recombinant proteins. Among them, Bacillus subtilis has been developed as an attractive host.

Vector map of pHT01

Vector map of pHT01

Vector map of pHT43

Vector map of pHT43

Features

  • It is non-pathogenic and is considered as a GRAS organism (generally regarded as safe)
  • It has no significant bias in codon usage
  • It is capable of secreting functional extracellular proteins directly into the culture medium (at present, about 60% of the commercially available enzymes are produced by Bacillus species)
  • A large body of information concerning transcription, translation, protein folding and secretion mechanisms, genetic manipulation and large-scale fermentation has been acquired
  • Useful for the construction of metagenomic libraries
  • pHT43 successfully used for production of endotoxin-free antimicrobial peptide (Luan et al., 2014)

Description

  • But there are also two obstacles reducing the use of B. subtilis:
    (i) production of a number of extracellular proteases which recognize and degrade heterologous proteins
    (ii) stable vector plasmids.
    The first obstacle has been largely solved by the construction of protease-deficient strains like our strain WB800N.
    The second has been completely overcome by introducing plasmids using the theta-mode of replication such as those derived from the natural plasmids pAMβ1 and pBS72 (Jannière et al., 1990; Titok et al., 2003).

  • The construction and use of four different expression vectors based on the E. coli - B. subtilis shuttle vector pMTLBS72 exhibiting full structural stability was published in 2005 (Nguyen et al., 2005).
  • The two vectors pHT01 and pHT43 allow high-level expression of recombinant proteins within the cytoplasm, where pHT43 directs the recombinant proteins into the medium. Both vectors are based on the strong σA-dependent promoter preceding the groE operon of B. subtilis which has been converted into an efficiently controllable (IPTG-inducible) promoter by addition of the lac operator.
  • Derivatives of pHT01 are available either with a 8xHis tag (pHT08), a Strep tag (pHT09) or a c-Myc tag (pHT10).

B. subtilis Host Strains

  • The following Bacillus subtilis strains suitable as hosts for gene expression are available:
  • For intracellular expression:
  • 1012 wild type: leuA8 metB5 trpC2 hsdRM1 (commonly used)
  • 168 Marburg: trpC2

  • Suited for secretion vectors:
  • WB800N: nprE aprE epr bpr mpr::ble nprB::bsrΔvpr wprA::hyg cm::neo; NeoR
  • Please note that WB800N carries resistance to neomycin.
  • (All strains listed are able to form spores.)

Resources

  • The Bacillus subtilis centered wiki SubtiWiki: A community-curated consensual annotation that is continuously updated.
  • SubtiPathways is a model of B. subtilis metabolism and regulation in SBML/SBGN (Systems Biology Markup Language/ Graphical Notation).
  • A Generic Protocol for Intracellular Expression of Recombinant Proteins in Bacillus subtilis (PDF)

ORDER INFORMATION

  • For shipping and storage information please click on Order#.
Order# Description Amount Price Data Sheet
PBS001 pHT01 vector, lyophilized plasmid DNA 10 µg 442,00 PDF
PBS001C pHT01-bgaB control vector, lyophilized plasmid DNA, available only in combination with regular vector 10 µg 171,00 PDF
PBS002 pHT43 vector, lyophilized plasmid DNA 10 µg 442,00 PDF
PBS002C pHT43-amyQ control vector, lyophilized plasmid DNA, available only in combination with regular vector 10 µg 171,00 PDF
PBS003 pHT08 vector, lyophilized plasmid DNA 10 µg 442,00 PDF
PBS004 pHT09 vector, lyophilized plasmid DNA 10 µg 442,00 PDF
PBS005 pHT10 vector, lyophilized plasmid DNA 10 µg 442,00 PDF
PBS005C pHT10-gfp+ control vector, lyophilized plasmid DNA, available only in combination with regular vector 10 µg 171,00 PDF
PBS020 Bacillus subtilis strain 1012wt 1 mL 465,00 PDF
PBS021 Bacillus subtilis strain 168 Marburg 1 mL 350,00 PDF
PBS022 Bacillus subtilis strain WB800N (for secretion vectors) 1 mL 613,00 PDF
  • All prices are in EURO excl. VAT and shipping. For further pricing and order information please ask your local distributor.

DOWNLOADS

  • Download the Bacillus subtilis Protein Expression System Handbook. (PDF)

Vector maps and Sequences

  • Vector map of pHT01 (pic)
  • Vector map of pHT43 (pic)
  • Vector map of pHT08 (pic)
  • Vector map of pHT09 (pic)
  • Vector map of pHT10 (pic)

Literature

  • Aguilar PS, Cronan JE Jr, de Mendoza D. (1998), A Bacillus subtilis gene induced by cold shock encodes a membrane phospholipid desaturase. J Bacteriol. 1998 Apr;180(8):2194-200. Pubmed
  • Anagnostopoulos, C. and Spizizen, J. (1961). Requirements for transformation in Bacillus subtilis. J. Bacteriol. 81:741-746. Pubmed
  • Biver, S., Steels, S., Portetelle, D., Vandenbol, M. (2013) Bacillus subtilis as a tool for screening soil metagenomic libraries for antimicrobial activities. J Microbiol Biotechnol. 2013 Jun 28;23(6):850-5. Pubmed
  • Jannière, L., Bruand, C. and Ehrlich, S.D. (1990) Structurally stable Bacillus subtilis cloning vectors, Gene 87:53-61. Pubmed
  • Luan C, Zhang HW, Song de G, Xie YG, Feng J, Wang YZ (2014) Expressing antimicrobial peptide cathelicidin-BF in Bacillus subtilis using SUMO technology. Appl Microbiol Biotechnol. 2014 Apr;98(8):3651-58. Pubmed
  • Nguyen HD, Phan TT, Schumann W (2007) Expression Vectors for the Rapid Purification of Recombinant Proteins in Bacillus subtilis Curr Microbiol. Vol. 55 (2007), pp. 89–93 Pubmed
  • Nguyen, D.H., Nguyen, Q.A., Ferreira, R.C., Ferreira, L.C.S., Tran, L.T. and Schumann, W. (2005). Construction of plasmid-based expression vectors for Bacillus subtilis. Plasmid; 2005 Nov; 54(3): 241-8. Pubmed
  • Phan, T.T.P., Nguyen, H.D. and Schumann, W. (2006). Novel plasmid-based expression vectorsfor intra- and extracellularproduction of recombinantproteins in Bacillus subtilis. Protein Expr. Purif.; 2006 Apr; 46(2): 189-95.. Pubmed
  • Thuy Le, A.T., Schumann W. (2007). A novel cold-inducible expression system for Bacillus subtilis. Protein Expr Purif. 2007 Jun;53(2):264-9. Epub 2007 Jan 9. Pubmed
  • Titok, M.A., Chapuis, J., Selezneva, Y.V., Lagodich, A.V., Prokulevich, V.A., Ehrlich, S.D. and Jannière, L. (2003). Bacillus subtilis soil isolates: plasmid replicon analysis and construction of a new theta-replicating vector, Plasmid 49: 53-62. Pubmed
  • Weber, M.H.W., Marahiel, M.A. (2003), Bacterial cold shock responses Sci Prog. 2003;86(Pt 1-2):9-75. Pubmed