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Bacillus subtilis Expression System | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Gram-positive bacteria are well known for their contributions to agricultural, medical and food biotechnology and for the production of recombinant proteins.
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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. Derivatives of pHT01 are available either with a 8xHis tag (pHT08), a Strep tag (pHT09) or a c-Myc tag (pHT10). | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
pAL12 – a cold-inducible VectorA further expression vector was constructed containing the cold-inducible des promoter of Bacillus subtilis. pAL12 allows for extracellular synthesis of recombinant proteins without the need for a cost-intensive inductor. 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 (Weber and Marahiel, 2003). In Bacillus subtilis, one of these cold-shock proteins is a membrane-bound desaturase (∆5-Des) encoded by the des gene (Aguilar et al., 1998). This enzyme catalyzes the introduction of a cis double bond at the ∆5 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 h (Thuy Le and Schumann 2007). | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Bacillus subtilis host strainsThe 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) Suited for secretion vectors: WB800N: nprE aprE epr bpr mpr :: ble nprB :: bsr .vpr wprA :: hyg cm :: neo; NeoR
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