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Bacillus subtilis Expression System

Bacillus subtilis Expression System
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Vector map pHT01; For a larger vector map please klick on the pictures

Vector map pHT43; For a larger vector map please klick on the pictures

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).

pAL12 – a cold-inducible Vector

A 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 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)

Resources

The Bacillus subtilis centred 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).

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Bacillus subtilis Expression Systems
	Introduction
	Order Information
	Downloads
	Literature

Please click on the respective order # for further details.

ORDER INFORMATION, SHIPPING & STORAGE:
order#	description	amount	Vector Map	Sequence
PBS001	pHT01 Bacillus subtilis expression vector, lyophilized plasmid DNA	10 μg	map	txt
PBS002	pHT43 Bacillus subtilis expression vector, lyophilized plasmid DNA	10 μg	map	txt
PBS003	pHT08 Bacillus subtilis expression vector, lyophilized plasmid DNA	10 μg	see pHT01	txt
PBS004	pHT09 Bacillus subtilis expression vector, lyophilized plasmid DNA	10 μg	see pHT01	txt
PBS005	pHT10 Bacillus subtilis expression vector, lyophilized plasmid DNA	10 μg	see pHT01	txt
PBS007	pAL12, Bacillus subtilis cold-inducible secretion expression vector	10 μg	map	txt
PBS020*	Bacillus subtilis strain 1012wt	1 ml
PBS021*	Bacillus subtilis strain 168 Marburg	1 ml
PBS022*	Bacillus subtilis strain WB800N (for secretion vectors)	1 ml
Shipped at room temperature (RT). Lyophilized plasmid DNA can be stored at 4°C. Once the DNA has been dissolved in sterile water or buffer we recommend storage at -20°C. *Strains are shipped on dry ice. For long-term storage store at -80°C.

Download the Bacillus subtilis Protein Expression System Handbook (PDF, 605 kb, Update 2010)

For further Vector System products please download the Vector Systems brochure (PDF, 3,9 MB)

Bacillus subtilis Expression System
	Introduction
	Order Information
	Downloads
	Literature

Literature

Anagnostopoulos, C. and Spizizen, J. (1961). Requirements for transformation in Bacillus subtilis.
J. Bacteriol. 81:741-746. Pubmed
Jannière, L., Bruand, C. and Ehrlich, S.D. (1990). Structurally stable Bacillus subtilis cloning vectors,
Gene 87:53-61. 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
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
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
Weber, M.H.W., Marahiel, M.A. (2003), Bacterial cold shock responses
Sci Prog. 2003;86(Pt 1-2):9-75. Pubmed
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