Innovative Tools for Molecular and Cell Biology

Innovative Tools for Molecular and Cell Biology

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Bacillus megaterium Expression System

Bacillus megaterium Expression System

Vector map of pN-Strep-Xa1622

Vector map of pN-Strep-Xa1622

Vector map of pHIS1525

Vector map of pHIS1525

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 BsrGI site*)
  • Versatile production (intracellular or extracellular)
  • Versatile purification (native, 6xHis-tag, Strep-tag, Strep/6xHis double-tag)
  • Removable tag versions (for TEV or Xa proteases) available

*BsrGI 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 to 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.
Strain YYBm1 is also a deletion mutant of wild type strain DSM319 (ΔnprM, ΔxylA) and thus unable to utilize xylose as carbon source.

Protoplast strain specifications:

Strain Features Literature
WH320 LacZ- Rygus et al. 1991
MS941 ΔnprM Wittchen and Meinhardt 1995
YYBm1 ΔnprM, ΔxylA Yang et al. 2006
Protein overexpression with Bacillus megaterium

Time dependence of induced expression of the enzymes Gdh (glucose dehydrogenase) and Mro (mutarotase) in B. megaterium. Enzymatic activity given in U/mg protein.

Bacillus megaterium and Escherichia coli

Electron microscope image of Bacillus megaterium and Escherichia coli, Dr. Manfred Rohde, Helmholtz-Center for Infection Research (HZI), Braunschweig, Germany

Examples

Selected proteins successfully overproduced in Bacilli strains with our B. megaterium vectors:

  • β-Galactosidase (LacZ)
  • Catabolite control protein (CcpA)
  • Clostridium difficile toxin A
  • Cobaltochelatase (CbiX)
  • Dextransucrase
  • Endolevanase (LevB)
  • Glucose dehydrogenase (GdhA)
  • Heat shock protein (HPr) from PTS (phosphotransferase sugar
    transport system)
  • Human single-chain urokinase-like plasminogen activator (rscuPA)
  • Levansucrase
  • Mutarotase (Mro)
  • Neopullulanase
  • Translocation ATPase of the preprotein translocase (SecA)
  • Trehalose repressor (TreR)

ORDER INFORMATION

  • For shipping and storage information please click on Order#.
Order# Description Amount Price Data Sheet
BMEG02 Bacillus megaterium protoplasts, strain WH320 5x500 µl 417,00 PDF
BMEG04 Bacillus megaterium protoplasts, strain YYBm1 5x500 µl 480,00 PDF
BMEG50 Bacillus megaterium protoplasts, strain MS941 5x500 µl 464,00 PDF
BMEG03 Bacillus megaterium vector pWH1520, lyophilized DNA 5 µg 270,00 PDF
BMEG10 Bacillus megaterium vector, pMM1522, lyophilized DNA 10 µg 270,00 PDF
BMEG11 Bacillus megaterium vector, pMM1525, lyophilized DNA 10 µg 270,00 PDF
BMEG12 Bacillus megaterium vector, pHIS1522, lyophilized DNA 10 µg 270,00 PDF
BMEG13 Bacillus megaterium vector, pHIS1525, lyophilized DNA 10 µg 270,00 PDF
BMEG14 Bacillus megaterium vector, pSTREP1525 10 µg 310,00 PDF
BMEG15 Bacillus megaterium vector pSTREPHIS1525 10 µg 310,00 PDF
BMEG20 Bacillus megaterium vector pC-His1622 10 µg 376,00 PDF
BMEG21 Bacillus megaterium vector pC-Strep1622 10 µg 463,00 PDF
BMEG22 Bacillus megaterium vector pN-His-TEV1622 10 µg 376,00 PDF
BMEG23 Bacillus megaterium vector pN-Strep-TEV1622 10 µg 463,00 PDF
BMEG24 Bacillus megaterium vector pN-StrepXa1622 10 µg 463,00 PDF
BMEG25 Bacillus megaterium vector, pSTOP1622, lyophilized DNA. 10 µg 376,00 PDF
PEC04 E. coli PxylA repressing vector, pMMEc4, lyophilized DNA 10 µg 215,00 PDF
  • All prices are in EURO excl. VAT and shipping. For further pricing and order information please ask your local distributor.

  • 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

  • Please note that these vectors are available only in combination with a regular B. megaterium expression vector! For background information regarding the controls please see Malten et al. 2006.
  • For your convenience and in order to provide a positive control, the following vectors validated for recombinant production and one-step affinity purification of L. reuteri levansucrase from growth medium using a B. megaterium expression system are available.
    • Order# Secretion vector description Amount Price Data Sheet
      BMEG10C pGFP1522, GFP expression vector, positive control 10 µg 59,00 PDF
      BMEG13C Levansucrase expression positive control, HIS-Tag 10 µg 59,00 PDF
      BMEG14C Levansucrase expression positive control, STREP-Tag 10 µg 59,00 PDF
      BMEG15C Levansucrase expression positive control, STREPHIS-Tag 10 µg 59,00 PDF
  • All prices are in EURO excl. VAT and shipping. For further pricing and order information please ask your local distributor.

Downloads

  • Get our B. megaterium Flyer as a short summary of our B. megaterium expression system. (PDF)
  • Bacillus megaterium Protein Production System Handbook (PDF)
  • Commentary – A short story about a big magic bug (Link)

Vector maps and Sequences

Vector map of pWH1520 (pic)
Vector map of pMM1522 (pic)
Vector map of pMM1525 (pic)
Vector map of pHIS1522 (pic)
Vector map of pHIS1525 (pic)
Vector map of pSTREP1525 (pic)
Vector map of pSTREPHIS1525 (pic)
Vector map of pC-His1622 (pic)
Vector map of pC-Strep1622 (pic)
Vector map of pN-His-TEV1622 (pic)
Vector map of pN-Strep-TEV1622 (pic)
Vector map of pN-StrepXa1622 (pic)
Vector map of pSTOP1622(pic)
Vector map of pMGBm19 (pic)

Sequence of pWH1520 (txt)
Sequence of pMM1522 (txt)
Sequence of pMM1525 (txt)
Sequence of pHIS1522 (txt)
Sequence of pHIS1525 (txt)
Sequence of pSTREP1525 (txt)
Sequence of pSTREPHIS1525 (txt)
Sequence of pC-His1622 (txt)
Sequence of pC-Strep1622 (txt)
Sequence of pN-His-TEV1622 (txt)
Sequence of pN-Strep-TEV1622 (txt)
Sequence of pN-StrepXa1622 (txt)
Sequence of pSTOP1622 (txt)
Sequence of pMGBm19 (txt)

Literature

  • Yang Y, Biedendieck R, Wang W, Gamer M, Malten M, Jahn D & Deckwer WD (2006) High yield recombinant penicillin G amidase production and export into the growth medium using Bacillus megaterium. Microb Cell Fact. 5:36. Pubmed
  • 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
  • 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
  • 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
  • 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
  • Wittchen KD, Meinhardt F (1995) Inactivation of the major extracellular protease from Bacillus megaterium DSM319 by gene replacement. Appl Microbiol Biotechnol 42:871–877 Pubmed
  • Vary PS.: Prime time for Bacillus megaterium Microbiology, May 1994; 140: 1001. Pubmed
  • 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
  • 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.
  • Saxena A, Zhang RW, Bollag JM.: Microorganisms capable of metabolizing the herbicide metolachlor. Appl. Environ. Microbiol. (1987) 53, 390-396. Pubmed Free
  • 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

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. 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
  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. 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
  4. 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
  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. 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
  7. 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
  8. 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
  9. 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
  10. 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
  11. 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
  12. 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