Description

Brevibacillus choshinensis, a gram positive bacterium, has excellent ability to produce many kinds of proteins extracellularly¹⁾. And the Brevibacillus Expression System based on this bacterium is well-suited for secretory production of heterologous proteins with high efficiency. Making use of this characteristic of the host bacterium, many successful records have been accumulated so far in this regard. This system has following merits:

• The host bacterium secretes proteins very efficiently.
• The host bacterium produces negligible amout of extracellular protease so that products remain unscathed in the culture medium.
• Proteins are produced as active forms.
• Easy to culture and sterilize the host bacterium.
• The host bacterium is amenable to genetic engineering.
• The host bacterium is guaranteed to be safe organism.

A part of our successful results are listed in Table 1. They cover enzymes, antigens, cytokines. All of them were produced at very high level and confirmed to have native biological activity. In addition to them, proteins from whole gamut of life forms, eubacteria, archaebacteria, eukaryotes, viruses, were successfully produced.
Special merit of this sytem is that it exhibits very high efficiency in the production of eukaryotic secretory proteins. Usually they have some cystein residues intramolecularly and disulfide bonds must be formed at exact positions precisely, which makes it difficult to achieve efficient production using other bacterial expression systems based on intracellular expression. Meanwhile, this system is very good at secretory production of proteins with native structure irrespective of whether they have disulfide bonds or not.

Table 1. Some examples of successful production of heterologous proteins using B. choshinensis host-vector system

Proteins	Origins	Production (g/L)	References
Enzymes
α-amylase	B. licheniformis	3.7
Sphingomyelinase	B. cereus	3.0
Xylanase	B. halodurans	0.2
CGTase	B. macerans	1.5	2)
Chitosanase	B. circulans	1.4
Hyper thermo-stable protease	A. pernix	0.1
Hyper thermo-stable nuclease	P. horikoshii	0.7
PDI	human	1.0	3)
Antigens
Surface antigen	E. rhusiopathiae	0.9
Surface antigen	T. pallidum	0.8
Cytokines
EGF	human	1.5	4)
IL-2	human	0.6	5)
NGF	mouse	0.2
IFN-γ	chicken	0.5	6)
TNF-α	bovine	0.4
GM-CSF	bovine	0.2
GH	flounder	0.2

The host bacterium exhibits high transformation efficiency by electroporation so that it is easy to construct expression clones. There is a shuttle vector between B. choshinensis and E. coli as well as the one maintained only in B. choshinensis. Thus, either the 2 step construction method using the shuttle vector or 1 step one using the vector for only B. choshinensis can be selected.
Two kinds of culture media are recommended for the culture of transformants. Cultures are performed using test tubes or flasks fixed on an appropriate shaker. After culture, supernatant is separated from cells by centrifugation from which target protein can be purified. The unnecessity to conduct cell disruption makes the purification procedure simpler and easier.

Components

Brevibacillus Expression System (Cat.#HB100)

• Expression vector
  pNY326 DNA 10 µg (Cat.#HB111)
  pNCMO2 DNA 10 µg (Cat.#HB112)
• Control vector
  pNCMO2BLA DNA 1 µg (Cat.#HB113)
• Competent cells (for electroporation)
  Brevibacillus choshinensis Electro-Cells 100 µl×10 tubes (Cat.#HB115)

Storage

Expression vectors pNY326, pNCMO2 DNA: -20°C
Control plasmid pNCMO2BLA DNA: -20°C
Competent cells Brevibacillus choshinensis Electro-Cells: -80°C
His-Tag Fusion Expression Vectors pNC-HisT, pNC-HisF, pNC-HisE DNA: -20°C

Sequence data

The Sequences of vectors are compressed into ZIP format. They are expanded with StuffIt Expander (for Macintosh), WinZip (for Windows).

Overview of Brevibacillus expression system

1. (1) pNCMO2
   pNCMO2 is a shuttle vector between B. choshinensis and E. coli. At first, expression plasmids can be constructed by emp loying E. coli as the host, then transfered into B. choshinensis cells.
   The pNCMO2 contains P2 promoter, which is one of 5 promoters driving transcription of cell wall protein (HWP) gene and does not work in E. coli.
   Therefore, it is suitable for cloning genes of which expressions are stressful for E. coli. In B. choshinensis cells, P2 promoter works as very strong promoter, enabling efficient protein production.
   Because of the strong promoter activity of P2 promoter, it shows detrimental effect on the growth of the transformants. In that case, the use of pNY326 is recommended.
   

   Fig. 1. Map of pNCMO2

   Primer design ↓ Gene amplification by PCR, Cloning into pNCMO2 using E. coli ↓ Confirmation of the sequence ↓ Transformation into B. choshinensis ↓ Expression trial

   
   (2) pNY326
   pNY326 can replicate in B. choshinensis cells and its size (3.4 kb) is much smaller than that of pNCMO2 (5.2 kb). In addition, the promoter activity of the vector is substantially weaker than that of pNCMO2. Taken together, it can be maintained stably even if it expresses rather toxic proteins to the host cells. In some case, higher efficient production of protein can be achieved using this vector because of the more stable growth of the transformants. Even after repeated inoculation to scale up the culture, it still exhibits stable productivity.
   Thus, this vector is especially suited to culture on large scale. The vector can be maintained only in B. choshinensis so that only B. choshinensis can be used as the host. The construction of expression plasmid must be constructed in one step using B. choshinensis as the host. Because the transformation efficiency must be sufficiently high, the use of Brevibacillus choshinensis Electro-Cells (10⁵ transformants/µg DNA) is recommended.
   

   Fig. 2. Map of pNY326

   Primer design ↓ Gene amplification by PCR, Cloning into pNY326 using B. choshinensis ↓ Confirmation of the sequence ↓ Expression trial

2. Cloning into expression vector
   The expression vectors have secretion signal sequence which derives from B. choshinensis cell wall protein gene. DNA fragment encoding the target protein should be cloned into the MCS which is located at the 3' end of the signal sequence.
   Using two restriction sites, one is on the signal sequence and the other on MCS, the gene of interest can be cloned into the vector with correct orientation.
   
   Fig. 3. Construction of B. choshinensis expression plasmid
3. The transformation of B. choshinensis cells can be done by electroporation. Selection marker is neomycin resistance. When the shuttle vector, pNCMO2, is used in combination with E. coli host, ampicillin resistance can be used as the selection marker.
4. Pick the transformant harboring the expression plasmid and culture it in the liquid medium. After 48 - 64 hrs' culture, prepare culture supernatant by centrifugation of the culture broth. An aliquot of the supernatant should be subjected to SDS-PAGE analysis to confirm the production of the target protein. If the production is not sufficient, please try scaling-up. Large scale culture of B. choshinensis is not difficult.

Overview of His-Tag Fusion Expression Vectors

pNC-HisT DNA, pNC-HisF DNA, and pNC-HisE DNA are shuttle vectors between Brevibacillus and E. coli derived from partially altered pNCMO2 DNA (Cat.# HB112), a secretory expression vector. These vectors are constructed with insertions of a His-Tag sequence (6×His sequence) located downstream of the secretion signal and of a protease recognition sequence for tag removal. The same as with pNCMO2, expression plasmids are constructed in E. coli and then transformed into Brevibacillus to conduct of expression experiments.
The His-Tag sequence is followed by a thrombin sequence in pNC-HisT DNA, a Factor Xa sequence in pNC-HisF DNA, or an enterokinase sequence in pNC-HisE DNA, offering the option to select a vector that is optimal for the target protein.
Like pNCMO2, these vectors use a P2 promoter derived from the host cell wall protein as its expression promoter. This P2 promoter is useful for cloning target genes because it is a very powerful promoter in Brevibacillus while it does not work in E. coli, thereby allowing for an efficient protein production in Brevibacillus.
This potent promoter activity, nevertheless, may interfere with the growth of transformants. In such a case, it is recommended to use pNY326 DNA (Cat. #HB111), which has a weaker promoter activity and can be stably maintained in the host cell. (However, His-Tag cannot be inserted into the N-terminus.)

When pNC-His DNA is used as the expression vector, the target protein can be easily purified using TALON^® Metal Affinity Resin (Cat. #635501) or another histidine tagged protein purification resin. After purification, if needed, His-Tag can be removed by exposing the purified product to the treatment by a protease which correspond to the cleavage site; Enterokinase, Factor Xa, or Thrombin.

Brevibacillus Expression System related products were developed and manufactured by Higeta Shoyu Co.,Ltd. and sold by TAKARA BIO INC. The use of this product is allowed for academic research only.
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