说明书(bmeg_handbook.pdf)

Features:

• Stable, high-yield protein expression in Bacillus megaterium
   
• Ideal for both small and large-scale protein production
• Tightly regulated and efficiently inducible xylose operon (up to 350-fold)
• No alkaline proteases activity even up to 5 hours after induction
• No endotoxins observed
• Versatile cloning (extended polylinker, additional BsrG I site*)
• Versatile production (intracellular or extracellular)
• Versatile purification (native, 6xHis-tag, Strep-tag, Strep/6xHis double-tag)
• Removeable tag versions (for TEV or Xa proteases) available
• Compatible with all B. subtilis vectors

*BsrG I site is not included in all vectors.

The B. megaterium expression system provides a versatile and easy-to-handle tool for stable and high-yield protein production, both small and large scale.

B. megaterium has proven to be an excellent alternative host to E. coli for heterologous gene expression. Unlike other bacilli strains, proteolytic degradation by alkaline proteases is avoided. In addition, there are no endotoxins found in the cell wall.

High Protein Yield
As a result, protein yields are exceptionally high even if inexpensive substrates are used. For example, the proteins mutarotase (Mro) and glucose dehydrogenase (Gdh) have been accumulated up to 20% and 30% of the total soluble protein, respectively. Using the tightly regulated xylose operon, the genes were induced 130- to 350-fold without proteolysis.

Versatile System with a Wide Range of Vectors
MoBiTec provides a wide range of useful vectors for the B. megaterium system that are adaptable for most applications and protein purification methods. These include vectors carrying a secretion signal peptide sequence, a 6xHis-tag, a Strep-tag, and a Strep/6xHis double-tag. Additionally, all vectors contain the tightly regulated and highly inducible xylose operon promoter.

B. megaterium protoplasts specifically optimized for transformation
The protoplasts supplied by MoBiTec are from B. megaterium strain WH320 developed by Prof. Dr. W. Hillen at the Institute of Microbiology in Erlangen, Germany. These protoplasts are prepared according to an optimized protocol resulting in the highest transformation efficiencies.
For secretion vectors we offer protoplasts of strain MS941. Both strains are mutants of DSM319, where WH320 is a chemically mutant, while MS941 has a defined deletion in the gene of the major extracellular protease NprM.

Examples:

Selected Proteins successfully over-produced in Bacilli strains with our B. megaterium vectors:

• β-Galactosidase (LacZ)¹
• Catabolite control protein (CcpA)^2,3
• Clostridium difficile toxin A⁴
• Cobaltochelatase (CbiX)⁵
• Dextransucrase⁶
• Endolevanase (LevB)⁷
• Glucose dehydrogenase (GdhA)¹
• Heat shock protein (HPr) from PTS (phosphotransferase sugar transport system)⁸
• Human single-chain urokinase-like plasminogen activator (rscuPA)¹
• Levansucrase⁹
• Mutarotase (Mro)¹
• Neopullulanase¹⁰
• Translocation ATPase of the preprotein translocase (SecA)¹¹
• Trehalose repressor (TreR)¹²