Bacillus megaterium Expression Systems

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 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
  

Order Information

Bacillus megaterium Expression Systems
	Introduction
	Order Information
	Downloads
	Literature
	FAQ

ORDER INFORMATION, SHIPPING & STORAGE:
order#	description	amount	Vector Map	Sequence
BMEG02	Bacillus megaterium protoplasts ready for transformation (strain WH320;1 ) Material is sufficient for 4 transformations plus control experiment.	5 x 500 µl
BMEG50	Bacillus megaterium protoplasts, strain MS941	5 x 500 µl
BMEG03	pWH1520 shuttle vector, original; lyophilized DNA	5 µg	map	txt
BMEG10	pMM1522 shuttle vector, improved; lyophilized DNA	10 µg	map	txt
BMEG11	pMM1525 shuttle vector with signal sequence; lyophilized DNA	10 µg	see pMM1522	txt
BMEG12	pHIS1522 shuttle vector, 6xHis-tagged; lyophilized DNA precursor of BMEG20	10 µg	map	txt
BMEG13	pHIS1525 shuttle vector with signal sequence; 6xHis-tagged; lyophilized DNA	10 µg	see pHIS1522	txt
BMEG14	pSTREP1525 shuttle vector with signal sequence; Strep-tagged; lyophilized DNA	10 µg	see pHIS1522	txt
BMEG15	pSTREPHIS1525 shuttle vector with signal sequence; Strep/6xHis double-tagged; lyophilized DNA	10 µg	map	txt
BMEG20	pC-His1622 shuttle vector, C-term. 6xHis-tag; lyophilized DNA	10 µg	map	txt
BMEG21	pC-Strep1622 shuttle vector, C-term. Strep-tag; lyophilized DNA	10 µg	map	txt
BMEG24	pN-StrepXa1622 shuttle vector, N-term. Strep-tag; Xa site; lyophilized DNA	10 µg	map	txt
BMEG25	pSTOP1622, shuttle vector, lyophilized DNA	10 µg	map	txt
shipped at RT, protoplasts shipped on dry ice store lyophilized vectors at 4°C, reconstituted vectors at -20°C, protoplasts at -70°C All vectors are E. coli / B. megaterium shuttle vectors Please note that protoplasts of Bacillus megaterium have a limited shelf life of some months only, depending on the date of production. Therefore purchase and use of protoplasts must be thoroughly planned.

Control Vectors

Bacillus megaterium Expression Systems Introduction Order Information Downloads Literature FAQ
Electron microscope image of Bacillus megaterium and Escherichia coli, Dr. Manfred Rohde, Helmholtz-Center for Infection Research (HZI), Braunschweig, Germany

Downloads

See also chapter 4 of our online catalog.
Get our new B. megaterium Flyer as a short summary of our B. megaterium expression system. (pdf 234 kb)

Downloads the Bacillus megaterium Protein Expression System Handbook (PDF, 1.3 Mb, Update 07.2008)

For further Vector System products please download the Vector Systems brochure (PDF, 3,9 MB)

Commentary – A short story about a big magic bug

Bacillus megaterium Expression Systems
	Introduction
	Order Information
	Downloads
	Literature
	FAQ

Literature

• Rygus T, Hillen W. : Inducible high-level expression of heterologous genes in Bacillus megaterium using the regulatory elements of the xylose-utilization
  operon,
  Appl. Microbiol. Biotechnol. (1991), 35, 594-599. Pubmed
  
  
• Rygus T, Scheler A, Allmansberger R,Hillen W.: Molecular cloning, structure, promoters and regulatory elements for transcription of the Bacillus megaterium encoded regulon for xylose utilization.
  Arch Microbiol, Jan 1991; 155(6): 535-42. Pubmed
  
  
• Hueck CJ, Kraus A, Schmiedel D, Hillen W.: Cloning, expression and functional analyses of the catabolite control protein CcpA from Bacillus megaterium.
  Mol Microbiol, Jun 1995; 16(5): 855-64. Pubmed
  
  
• Vary PS.: Prime time for Bacillus megaterium
  Microbiology, May 1994; 140: 1001. Pubmed
  
  
• Saxena A, Zhang RW, Bollag JM.: Microorganisms capable of metabolizing the herbicide metolachlor.
  Appl. Environ. Microbiol. (1987) 53, 390-396. Pubmed Free
  
  
• Selvanayagam, M. and Vijaya, J., Degradation of persistent insecticides by aquatic bacteria as sole source of carbon.
  J. Environ. Biol. (1989) 10, 399-407.
  
  
• Meinhardt F, Stahl U, Ebeling W. Highly efficient expression of homologous and heterologous genes in Bacillus megaterium.
  Appl. Microbiol. Biotechnol. (1989) 30, 343-350.PUYET A. (1) ; SANDOVAL H. ; LOPEZ P. ; AGUILAR A. ; MARTIN J. F. ; ESPINOSA M.
  
  
• Puyet A, Sandoval H, Lopez P, Aguilar A, Martin JF, Espinosa M.: A simple medium for rapid regeneration of Bacillus subtilis protoplasts transformed with plasmid DNA.
  FEMS Microbiol. Lett. (1987) 40, 1-5.
  
  
• Laemmli, U. K.: Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
  Nature (1970) 227, 680-685 Pubmed
  
  
• Antelmann, H., Tjalsma, H., Voigt, B., Ohlmeier, S., Bron, S., van Dijl, J.M. and Hecker, M. : A proteomic view on genome-based signal peptide predictions.
  Genome Res. 2001 Sep;11(9):1484-502. Pubmed Free
  
  
• Malten, M., Biedendieck, R., Gamer, M., Drews, A.-C., Stammen, S., Buchholz, K., Dijkhuizen, L. and Jahn, D.: A Bacillus megaterium plasmid system for the production, export and one-step purification of affinity tagged heterologous levansucrase from the growth medium.
  Appl Environ Microbiol. 2006 Feb;72(2):1677-9. Pubmed Free
  
  
• Wang W, Hollmann R, Deckwer WD.: Comparative proteomic analysis of high cell density cultivations with two recombinant Bacillus megaterium strains for the production of a heterologous dextransucrase.
  Proteome Sci. 2006 Oct 5;4:19. Pubmed Free
  

Potential industrial and diagnostical applications:

• Vary, P. S. (1992) Development of genetic engineering in Bacillus megaterium : an example of the versatility and potential of industrially important bacilli
  Biotechnology, Jan 1992; 22: 251-310. Pubmed
  
  
• Ginsburgh, C., Spaulding, D., Robey, G., Shivakumar, A.M.O., Vanags, R., Katz, L., Fox, J.L. (1989): Sporulation promoter spoVG controlled expression of PP42 gene of HIV-1 in Bacillus megaterium. Abstract from an International Conference on AIDS, Montreal.

successfully expressed proteins with our B. megaterium vectors:

1. Rygus T, Hillen W. : Inducible high-level expression of heterologous genes in Bacillus megaterium using the regulatory elements of the xylose-utilization operon,
   Appl. Microbiol. Biotechnol. (1991), 35, 594-599. Pubmed
   
   
2. Seidel G, Diel M, Fuchsbauer N, Hillen W.:
   Quantitative interdependence of coeffectors, CcpA and cre in carbon catabolite regulation of Bacillus subtilis
   FEBS J., May 2005; 272: 2566 - 2577. Pubmed Free
   
   
3. Hueck CJ, Kraus A, Schmiedel D, Hillen W.: Cloning expression and functional analyses of the catabolite control protein ccpA from Bacillus megaterium,
   Mol. Microbiol. (1995) 16(5), 855-864. Pubmed
   
   
4. Burger S, Tatge H, Hofmann F, Genth H, Just I, Gerhard R.: Expression of recombinant Clostridium difficile toxin A using the Bacillus megaterium system.
   Biochem Biophys Res Commun. 2003 Aug 1;307(3):584-8. Pubmed Free
   
   
5. Leech HK, Raux E, McLean KJ, Munro AW, Robinson NJ, Borrelly GP, Malten M, Jahn D, Rigby SE, Heathcote P, Warren MJ.: Characterization of the cobaltochelatase CbiXL: evidence for a 4Fe-4S center housed within an MXCXXC motif.
   J. Biol. Chem., Oct 2003; 278: 41900 - 41907. Pubmed Free
   
   
6. Wang W, Hollmann R, Furch T, Nimtz M, Malten M, Jahn D, Deckwer WD.: Proteome analysis of a recombinant Bacillus megaterium strain during heterologous production of a glucosyltransferase
   Proteome Sci. 2005; 3: 4. Pubmed Free
   
   
7. Daguer JP, Geissmann T, Petit-Glatron MF, Chambert R.: Autogenous modulation of the Bacillus subtilis sacB–levB–yveA levansucrase operon by the levB transcript
   Microbiology, Nov 2004; 150: 3669 - 3679. Pubmed Free
   
   
8. Kraus A, Kuster E, Wagner A, Hoffmann K, Hillen W.: Identification of a co-repressor binding site in catabolite control protein CcpA.
   Mol Microbiol, Dec 1998; 30(5): 955-63. Pubmed Free
   
   
9. Malten, M., Biedendieck, R., Gamer, M., Drews, A.-C., Stammen, S., Buchholz, K., Dijkhuizen, L. and Jahn, D.: A Bacillus megaterium plasmid system for the production, export and one-step purification of affinity tagged heterologous levansucrase from the growth medium.
   Appl Environ Microbiol. 2006 Feb;72(2):1677-9. Pubmed Free
   
   
10. Kamasaka H, Sugimoto K, Takata H, Nishimura T, Kuriki T.: Bacillus stearothermophilus Neopullulanase Selective Hydrolysis of Amylose to Maltose in the Presence of Amylopectin
    Appl. Envir. Microbiol., Apr 2002; 68: 1658 - 1664. Pubmed Free
    
    
11. Leloup L, Driessen AJ, Freudl R, Chambert R, Petit-Glatron MF.: Differential Dependence of Levansucrase and -Amylase Secretion on SecA (Div) during the Exponential Phase of Growth of Bacillus subtilis
    J. Bacteriol., Mar 1999; 181: 1820 - 1826. Pubmed Free
    
    
12. Burklen L, Schock F, Dahl MK.: Molecular analysis of the interaction between the Bacillus subtilis trehalose repressor TreR and the tre operator.
    Mol Gen Genet, Oct 1998; 260(1): 48-55. Pubmed