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Products >  Protein_Expression_and_Purification >  Bacterial_Expression_Systems >  High_Yield_Expression >  Brevibacillus_Ligation-independent_cloning

Secreted protein production in Brevibacillus choshinensis: Ligation-independent cloning

Brevibacillus choshinensis is a highly effective alternative bacterial recombinant protein expression system and is particularly advantageous for expressing secreted protein products. This gram-positive bacterial system can allow production of micrograms to grams of secreted protein per L of culture. Advantages of Brevibacillus choshinensis as an expression system include lack of endogenous proteases and endotoxin, simple culturing methods, ability to secrete large amounts of recombinant protein, and ease of handling. The BIC (Brevibacillus in vivo Cloning) kit allows ligation-independent cloning, making secreted protein production projects quick and easy. Because subcloning steps are avoided, this ligation-independent cloning method is suitable for generating recombinant proteins that are toxic to E. coli. Vectors included in this system allow inclusion of a His-tag sequence to permit  purification of secreted protein from culture media. In addition to facilitating secreted protein production, the BIC system allows construction of plasmids for intracellular expression that lack the secretion signal or plasmids for direct secretory expression that lack the His-Tag sequence.

The BIC method for use with Brevibacillus choshinensis allows PCR product to be mixed with pBIC Vector DNA. Both insert and vector are designed to include 15-bp homologous overhangs to allow homologous recombination in vivo, yielding the desired construct. Four different pBIC vectors are included, each with a different type of secretion signal sequence. This allows rapid generation of a suite of constructs to allow screening for the expression plasmid that allows optimal secretory expression. The pBIC system enables creation of three different types of constructs for Brevibacillus choshinensis expression: 1) His-tag secreted protein expression, 2) untagged secreted protein expression, and 3) intracellular protein expression.

  At-A-Glance   Documents   Images & Data

Features

  • Allows efficient production of secreted protein in Brevibacillus choshinensis
  • Includes supplies for ligation-independent cloning: pBIC linear vectors, control insert, and Brevibacillus choshinensis competent cells
  • Allows generation of three different types of constructs for tagged or untagged secreted protein expression or intracellular expression
  • Suite of four pBIC vectors allow screening for the optimal secretion signal sequence for target protein of interest

Applications

  • Production of recombinant protein (either secreted or intracellular)

Components

Brevibacillus Expression System - BIC System (Cat. # HB300)

Component
Amount
pBIC DNA Set (Cat. #HB310), 10 rxns
Linear Vector DNA
pBIC1 DNA
5 µg (100 ng/µL)
pBIC2 DNA
5 µg (100 ng/µL)
pBIC3 DNA
5 µg (100 ng/µL)
pBIC4 DNA
5 µg (100 ng/µL)
Control (Insert DNA)
BLA
1 µg (80 ng/µL)
Brevibacillus Competent Cells (Cat. #HB116)
Brevibacillus Competent Cells100 µL x 10
MT medium1 mL x 10
Solution A1 mL x 1
Solution B1 mL x 2

Storage

Store pBIC DNA Set components at -20°C. Store Brevibacillus Competent Cells, MT medium, Solution A, and Solution B at -80°C.

pBIC Vector DNA

Promoter
Portion of the 5′ sequence upstream of the B. choshinensis P22 gene, which provides strong constitutive expression
Secretion Signal
pBIC1: Secretion signal for BbrPI from B. choshinensis
pBIC2: Secretion signal for a cell wall protein from B. brevis
pBIC3: Secretion signal for the P22 gene from B. choshinensis
pBIC4: Modified secretion signal for pBIC2
Terminator
46 bp termination signal inserted downstream from the cloning site
Rep
Encodes a protein related to plasmid replication (derived from pUB110)
Ori
Plasmid replication origin (derived from pUB110)
NmRNeomycin resistance gene (selection marker)

Ligation-independent Cloning Method

The ligation-independent cloning method enabled by the pBIC system involves three easy steps: 1) PCR to generate insert, 2) mixing of insert and vector and transformation of Brevibacillus Competent Cells, and 3) plating on media containing neomycin. Download the product User Manual for details.


 
 
Products
Cat. # Product Contents Size Price License Units Select
HB116 ◊Brevibacillus Choshinensis Competent Cells 10 x 100 uL $359.00 License Statements
HB310 pBIC DNA Set 1 Kit $955.00 License Statements
HB300 BIC System Each $1,300.00 License Statements
 

 

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