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B. subtilis Protein Expression

BSUB VECTORS - Bacillus subtilis vectors, 10μg

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Bacillus subtilis vectors

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 because of several reasons:

  • 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.

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 (Janničre et al., 1990; Titok et al., 2003).

Quite recently, 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 (Nguyen et al., 2005).

The new vector pHT01 allows high-level expression of recombinant proteins within the cytoplasm, whereas pHT43 secretes the recombinant proteins into the medium.

Derivatives of pHT01 are available either with a 8xHis tag (pHT08), a Strep tag (pHT09) or a c-Myc tag (pHT10).

pHT01 pHT43


The 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.

The pAL Vectors

Two further expression vectors were constructed both containing the cold-inducible des promoter of Bacillus subtilis, where pAL10 allows for intra- and pAL12 for extracellular synthesis of recombinant proteins. When mid-exponential phase bacterial cells are rapidly transferred from 37°C to 25°C or even a lower temperature, the synthesis of most cellular proteins greatly decreases, while that of cold-shock proteins is transiently upregulated. In Bacillus subtilis, one of these cold-shock proteins is a membrane-bound desaturase (Δ5-Des) encoded by the des gene. This enzyme catalyzes the introduction of a cis double bond at the Δ5 position of a wide variety of saturated fatty acids. It has been shown that the des gene is tightly regulated during cold shock. Production of recombinant proteins started within the first 30 min after temperature downshock to 25°C and continued for about 5 hr.

Summary of available B. Subtilis vectors:

VectorCat. #TagSecretion
pHT01PBS001NONO (intracellular)
pHT43PBS002NOYES (extracellular)
pHT08PBS0038 X HISNO
pHT09PBS004Strep TagNO
pHT10PBS005c-Myc TagNO
pAL10PBS006NO (cold inducible)NO
pAL12PBS007NO (cold inducible)YES

 

Bacillus subtilis User Manual