Production of Antibody Fragments in the Gram-Positive Bacterium Bacillus megaterium

  • Miriam SteinwandEmail author
  • Eva Jordan
  • Michael Hust
Part of the Springer Protocols Handbooks book series (SPH)


For many applications antibody fragments like scFv or Fab are an alternative to full size IgGs. These fragments can be produced in bacterial host systems, which are less time and cost intensive than mammalian cell culture, the production host for more complex IgGs. The Gram-negative bacterium Escherichia coli has been employed for the production of recombinant antibody fragments for over 20 years, but certain disadvantages could not be eliminated, such as its inefficient secretory capacity due to its outer membrane. The Gram-positive, apathogenic soil bacterium Bacillus megaterium has been studied for the secretion of antibody fragments directly into the culture medium, thereby easing the downstream processing. Here, we describe the production of antibody fragments in B. megaterium, from preparation of protoplasts for transformation to purification of the recombinant protein by IMAC


Antibody Fragment Elution Fraction Lysozyme Solution scFv Fragment Produce Alkaline Protease 
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  1. Barg H, Malten M, Jahn M, Jahn D (2005). Protein and vitamin production in Bacillus megaterium. In Methods in Biotechnology – Microbial products and biotransformations. Humana, New JerseyGoogle Scholar
  2. Biedendieck R, Beine R, Gamer M, Jordan E, Buchholz K, Seibel J, Dijkhuizen L, Malten M, Jahn D (2007) Export, purification and activities of affinity tagged Lactobacillus reuteri levansucrase produced by Bacillus megaterium. Appl Microbiol Biotechnol 74:1062–1073PubMedCrossRefGoogle Scholar
  3. Hust M, Dübel S (2004) Mating antibody phage display to proteomics. Trends Biotechnol 22:8–14PubMedCrossRefGoogle Scholar
  4. Inoue Y, Ohta T, Tada H, Iwasa S, Udaka S, Yamagata H (1997) Efficient production of a functional mouse/human chimeric Fab’ against human urokinase- type plasminogen activator by Bacillus brevis. Appl Microbiol Biotechnol 48:487–492PubMedCrossRefGoogle Scholar
  5. Jordan E, Hust M, Roth A, Biedendieck R, Schirrmann T, Jahn D, Dübel S (2007a) Production of recombinant antibody fragments in Bacillus megaterium. Microb Cell Fact 6:2PubMedCrossRefGoogle Scholar
  6. Jordan E, Al-Halabi L, Schirrmann T, Hust M, Dübel S (2007b) Production of single chain Fab (scFab) fragments in Bacillus megaterium. Microb Cell Fact 6:38PubMedCrossRefGoogle Scholar
  7. Malten M, Hollmann R, Deckwer WD, Jahn D (2005) Production and secretion of recombinant Leuconostoc mesenteroides Dextransucrase DsrS in Bacillus megaterium. Biotechnol Bioeng 89:206–218PubMedCrossRefGoogle Scholar
  8. Malten M, Biedendieck R, Gamer M, Drews AC, Stammen S, Buchholz K, Dijkhuizen L, Jahn D (2006) A Bacillus megaterium plasmid system for the production, export, and one-step purification of affinity-tagged heterologous levansucrase from growth medium. Appl Environ Microbiol 72:1677–1679PubMedCrossRefGoogle Scholar
  9. Schirrmann T, Al-Halabi L, Dübel S, Hust M (2008) Production systems for recombinant antibodies. Frontiers of Bioscience 13:4576–4594CrossRefGoogle Scholar
  10. Shiroza T, Shinozaki-Kuwahara N, Hayakawa M, Shibata Y, Hashizume T, Fukushima K, Udaka S, Abiko Y (2003) Production of a single-chain variable fraction capable of inhibiting the Streptococcus mutans glucosyltransferase in Bacillus brevis: construction of a chimeric shuttle plasmid secreting its gene product. Biochim Biophys Acta 1626:57–64PubMedCrossRefGoogle Scholar
  11. Taussig MJ, Stoevesandt O, Borrebaeck C, Bradbury A, Dübel S, Frank R, Gibson T, Gold L, Herberg F, Hermjakob H, Hoheisel J, Joos T, Konthur Z, Landegren U, Plückthun A, Ueffing M, Uhlen M (2007) ProteomeBinders: Planning a European resource of affinity reagents for analysis of the human proteome. Nature Methods 4:13–17PubMedCrossRefGoogle Scholar
  12. Vary PS (1994) Prime time for Bacillus megaterium. Microbiology 140:1001–1013PubMedCrossRefGoogle Scholar
  13. Wittchen KD, Meinhardt F (1995) Interactivation of the major extracellular protease from Bacillus megaterium DSM319 by gene replacement. Appl Microbiol Biotechnol 42:817–877CrossRefGoogle Scholar
  14. Wu SC, Ye R, Wu XC, Ng SC, Wong SL (1998) Enhanced secretory production of a single-chain antibody fragment from Bacillus subtilis by coproduction of molecular chaperones. J Bacteriol 180:2830–2835PubMedGoogle Scholar
  15. Wu SC, Yeung JC, Duan Y, Ye R, Szarka SJ, Habibi HR, Wong SL (2002) Functional production and characterization of a fibrin-specific single-chain antibody fragment from Bacillus subtilis: effects of molecular chaperones and a wall-bound protease on antibody fragment production. Appl Environ Microbiol 68:3261–3269PubMedCrossRefGoogle Scholar
  16. 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 5:36PubMedCrossRefGoogle Scholar
  17. Yang Y, Malten M, Grote A, Jahn D, Deckwer WD (2007) Codon optimized Thermobifida fusca hydrolase secreted by Bacillus megaterium. Biotechnol Bioeng 96:780–794PubMedCrossRefGoogle Scholar

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© Springer-Verlag Berlin Heidelberg 2010

Authors and Affiliations

  1. 1.Department of Biotechnology Institute of Biochemistry and BiotechnologyTechnical University BraunschweigBraunschweigGermany

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