Description

Brevibacillus (Bacillus brevis) Expression System is an excellent and high-efficient protein production system. This bacterium, a Gram-positive microbe, is characterized in particular by its ability to secrete/produce large amount of proteins¹⁾. This characteristic has been successfully used in the secretory production of a large number of heterologous proteins.
Recent studies have shown that this bacterium is excellent not only in secretory productions but also in intracellular productions. Proteins that insolubilize and precipitate when produced by E. coli may be recovered in a soluble state when produced by this bacterium. This system is recommended for the production of functional proteins that are naturally produced intracellularly but become insolubilized and cannot undergo in vitro refolding when produced by E. coli. This system has the following features:

• efficient intracellular production of soluble heterologous proteins
• easy to culture and sterilize
• amenable to genetic manipulations
• a safe host

With high transformation efficiency by electroporation, this host is amenable to genetic manipulation. Using a shuttle vector between this bacterium and E. coli, expression vectors can be constructed in E. coli. A vector with a His-tag inserted to the N-terminus is also available. The target protein can be easily purified using a histidine tagged protein purification column. The His-Tag can be removed by exposing the purified protein to enterokinase treatment.

Components

• Intracellular expression vectors
  pNI DNA  10 µg (Cat.# HB131)
  pNI-His DNA  10 µg (Cat.# HB132)

Storage

-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 Intracellular expression vectors

1. Selection of expression vectors
   1. pNI DNA
      pNI DNA is an intracellular expression vector obtained by removing the secretory signal segment from the secretory expression vector pNCMO2 DNA (Cat.# HB112) to allow intracellular accumulation of products expressed. The rest of its basic structures are the same as those of pNCMO2 DNA. It is a shuttle vector between Brevibacillus and E. coli used to construct expression plasmids in host E. coli, which are then transferred into Brevibacillus for expression analysis.
      pNI DNA uses a P2 promoter for the host cell wall protein as its expression promoter. This P2 promoter exhibits a weak action in E. coli and is useful for cloning target genes. It is however a very powerful promoter in Brevibacillus and suitable for efficient protein productions in Brevibacillus.
   2. pNI-His DNA
      pNI-His DNA, which allows the simple purification of expressed products by nickel chelate resin, is an intracellular expression vector containing a His-tag sequence (6×His) and an enterokinase recognition sequence for tag removal. The rest of its structures are the same as those of pNI DNA.
   
   pNI DNA: 5,055 bp
   pNI-His DNA: 5,079 bp
   Fig. 1. Vector Map of pNI DNA
2. Cloning into expression vectors
   For pNI DNA, a gene of interest is inserted into the multiple cloning site (MCS). If the Nco I site is usable, extra amino acids derived from MCS would not be inserted into the produced protein. Using a restriction site other than Nco I will result in the addition of extra amino acids to its N-terminus, depending on the site.
   For pNI-His DNA, a gene of interest is inserted into a site downstream of BamH I.
3. Transformation of Brevibacillus
   The transformation of B. choshinensis cells can be done by electroporation. Selection marker is neomycin resistance. When the shuttle vector is used in combination with E. coli host, ampicillin resistance can be used as the selection marker.
4. Detection of protein production and scale-up
   Culture a negative control concurrently to confirm the expression of the target protein. The transformant harboring the expression plasmid for the target protein is cultured in the specified liquid medium with shaking for 48 to 64 hours to obtain the target protein. Subject the bacterial culture to SDS-PAGE analysis, or a similar test, to confirm the presence/absence of its expression. Scale up the culture level when a high productivity is required, because large scale culture of Brevibacillus is relatively easy.

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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