title: High-level intracellular expression of heterologous proteins in Brevibacillus choshinensis SP3 under the control of a xylose inducible promoter.
abstract: BACKGROUND
  In past years research has focused on the development of alternative Gram positive bacterial expression systems to produce industrially relevant proteins. Brevibacillus choshinensis is an easy to handle non-sporulating bacterium, lacking extracellular proteases, that has been already shown to provide a high level of recombinant protein expression. One major drawback, limiting the applicability of the Brevibacillus expression system, is the absence of expression vectors based on inducible promoters. Here we used the PxylA inducible promoter, commonly employed in other Bacillae expression systems, in Brevibacillus.
  
  
  RESULTS
  Using GFP, α-amylase and TcdA-GT as model proteins, high level of intracellular protein expression (up to 250 mg/L for the GFP) was achieved in Brevibacillus, using the pHis1522 vector carrying the B. megaterium xylose-inducible promoter (PxylA). The GFP expression yields were more than 25 fold higher than those reported for B. megaterium carrying the same vector. All the tested proteins show significant increment in their expression levels (2-10 folds) than those obtained using the available plasmids based on the P2 constitutive promoter.
  
  
  CONCLUSIONS
  Combining the components of two different commercially available Gram positive expression systems, such as Brevibacillus (from Takara Bio) and B. megaterium (from Mobitec), we demonstrate that vectors based on the B. megaterium PxylA xylose inducible promoter can be successfully used to induce high level of intracellular expression of heterologous proteins in Brevibacillus.
authors: Novartis Vaccines and Diagnostics, Via Fiorentina 1, 53100, Siena, Italy.
authors: Nunzia
authors: N
authors: D'Urzo
authors: Manuele
authors: M
authors: Martinelli
authors: Chiara
authors: C
authors: Nenci
authors: Cecilia
authors: C
authors: Brettoni
authors: John L
authors: JL
authors: Telford
authors: Domenico
authors: D
authors: Maione
mesh_terms: D019747:Aldose-Ketose Isomerases / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics; D001427:Bacterial Toxins / Q000235:genetics / Q000378:metabolism; D058675:Brevibacillus / Q000378:metabolism*; D004768:Enterotoxins / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014994:Xylose / Q000378:metabolism*; D000516:alpha-Amylases / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004768:Enterotoxins; D011994:Recombinant Proteins; C041348:tcdA protein, Clostridium difficile; D049452:Green Fluorescent Proteins; D014994:Xylose; D000516:alpha-Amylases; D019747:Aldose-Ketose Isomerases; C023298:xylose isomerase
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