Innovative Tools for Molecular and Cell Biology

Innovative Tools for Molecular and Cell Biology

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Bacillus subtilis Pgrac100 Expression Vectors

Highly Efficient Intracellular Production of Recombinant Proteins in Bacillus subtilis

Features

  • Strong promoter with improved regulatory elements
  • Enhanced amount of expressed recombinant proteins
  • Inducible protein expression
  • E. coli / B. subtilis shuttle vectors with different tags (His-tag, Strep-tag)
  • B. subtilis is considered as a GRAS organism (generally regarded as safe)

B. subtilis as Expression Host

  • Bacillus subtilis has been developed as an attractive host because of several reasons:
    1. It is non-pathogenic and is considered as a GRAS organism (generally regarded as safe)
    2. It has no significant bias in codon usage
    3. 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)
    4. A large body of information concerning transcription, translation, protein folding and secretion mechanisms, genetic manipulation and large-scale fermentation has been acquired.

  • 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, and (ii) stable vector plasmids. The first obstacle has been largely solved by the construction of protease-deficient strains. And 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 Pgrac100 Vectors

  • All Pgrac100 vectors use the strong promoter preceding the groESL operon of Bacillus subtilis with improved regulatory elements fused to the lac operator allowing their induction by IPTG. Nucleotides were optimized at the conserved regions of the groESL promoter including the UP element, the −35 and the −15 region. Combination of these changes into one promoter enhanced the amount of recombinant proteins accumulating intracellularly up to about 30% of the total cellular protein of B. subtilis (Phan et al., 2011). In addition, the target proteins could be also expressed efficiently in E. coli in some cases.

B. subtilis Host Strains

  • The following Bacillus subtilis strains suitable as hosts for gene expression are available:
  • For intracellular expression:
  • AS1: 1012 hrcA::neo (producing strain for enhancing solubility of intracellular protein) (Schulz and Schumann, 1996, and Phan et al., 2006)
  • 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.)
Vector map of pHT253 with N-terminal His-tag

Vector map of pHT253 with N-terminal His-tag

Vector map of pHT254 with C-terminal  His-tag

Vector map of pHT254 with C-terminal His-tag

Vector map of pHT255 with C-terminal Strep-tag

Vector map of pHT255 with C-terminal Strep-tag

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).

ORDER INFORMATION

  • For shipping and storage information please click on Order#.
Order# Description Amount Price Data Sheet
PBS013 pHT253 (pHT-Pgrac100-His-Tag-MCS) 10 µg 529,00 PDF
PBS014 pHT254 (pHT-Pgrac100-MCS-His-Tag) 10 µg 529,00 PDF
PBS015 pHT255 (pHT-Pgrac100-MCS-STREP-Tag) 10 µg 529,00 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
PBS026 Bacillus subtilis strain AS1 1 mL 512,00 PDF
  • All prices are in EURO excl. VAT and shipping. For further pricing and order information please ask your local distributor.

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Vector maps and Sequences

Vector map of pHT253 (pHT-Pgrac100-His-tag-MCS) (pic)
Vector map of pHT254 (pHT-Pgrac100-MCS-His-tag) (pic)
Vector map of pHT255 (pHT-Pgrac100-MCS-Strep-tag) (pic)

Sequence of pHT253 (pHT-Pgrac100-His-tag-MCS) (txt)
Sequence of pHT254 (pHT-Pgrac100-MCS-His-tag) (txt)
Sequence of pHT255 (pHT-Pgrac100-MCS-Strep-tag) (txt)

Literature

  • Jannière, L., Bruand, C. and Ehrlich, S.D. (1990). Structurally stable Bacillus subtilis cloning vectors. Gene 87:53-61. Pubmed

  • Phan, T.T.P., Nguyen, H.D. and Schumann, W. (2006). Novel plasmid-based expression vectors for intra- and extracellular production of recombinant proteins in Bacillus subtilis. Protein Expr. Purif. 46, 189–195. Pubmed

  • Phan, T.T.P., Nguyen, H.D. and Schumann, W. (2011). Development of a strong intracellular expression system for Bacillus subtilis by optimizing promoter elements. J. Biotechnology, 2012 Jan;157(1):167-72. Epub 2011 Nov 10. Pubmed

  • Phan, T.T.P., Tran, L.T., Schumann, W. and Nguyen, H.D. (2015). Development of Pgrac100-based expression vectors allowing high protein production levels in Bacillus subtilis and relatively low basal expression in Escherichia coli. Microbial Cell Factories 14:72 Pubmed

  • Schulz, A. and Schumann, W. (1996) hrcA, the first gene of the Bacillus subtilis dnaK operon encodes a negative regulator of class I heat shock genes. J.Bacteriol., 178, 1088-1093. 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