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 (Jannière 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 two new 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).
Bacillus subtilis host strains
The following Bacillus subtilis strains suitable as hosts for gene expression are available:
For intracellular expression we offer:
1012 wild type: leuA8 metB5 trpC2 hsdRM1 (commonly used)
168 Marburg: trpC2 (Trp - )
Suited for secretion vectors:
WB800N: nprE aprE epr bpr mpr :: ble nprB :: bsr .vpr wprA :: hyg cm :: neo; NeoR (Please note that WB800N carries resistance to neomycin)