Abstract
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
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
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 Jersey
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 74:1062–1073
Hust M, Dübel S (2004) Mating antibody phage display to proteomics. Trends Biotechnol 22:8–14
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 48:487–492
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 6:2
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 6:38
Malten M, Hollmann R, Deckwer WD, Jahn D (2005) Production and secretion of recombinant Leuconostoc mesenteroides Dextransucrase DsrS in Bacillus megaterium. Biotechnol Bioeng 89:206–218
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 72:1677–1679
Schirrmann T, Al-Halabi L, Dübel S, Hust M (2008) Production systems for recombinant antibodies. Frontiers of Bioscience 13:4576–4594
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 1626:57–64
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 4:13–17
Vary PS (1994) Prime time for Bacillus megaterium. Microbiology 140:1001–1013
Wittchen KD, Meinhardt F (1995) Interactivation of the major extracellular protease from Bacillus megaterium DSM319 by gene replacement. Appl Microbiol Biotechnol 42:817–877
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 180:2830–2835
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 68:3261–3269
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
Yang Y, Malten M, Grote A, Jahn D, Deckwer WD (2007) Codon optimized Thermobifida fusca hydrolase secreted by Bacillus megaterium. Biotechnol Bioeng 96:780–794
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2010 Springer-Verlag Berlin Heidelberg
About this protocol
Cite this protocol
Steinwand, M., Jordan, E., Hust, M. (2010). Production of Antibody Fragments in the Gram-Positive Bacterium Bacillus megaterium . In: Kontermann, R., Dübel, S. (eds) Antibody Engineering. Springer Protocols Handbooks. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-01147-4_23
Download citation
DOI: https://doi.org/10.1007/978-3-642-01147-4_23
Publisher Name: Springer, Berlin, Heidelberg
Print ISBN: 978-3-642-01146-7
Online ISBN: 978-3-642-01147-4
eBook Packages: Springer Protocols