title: Recombinant production of the antibody fragment D1.3 scFv with different Bacillus strains.
abstract: BACKGROUND
  Different strains of the genus Bacillus are versatile candidates for the industrial production and secretion of heterologous proteins. They can be cultivated quite easily, show high growth rates and are usually non-pathogenic and free of endo- and exotoxins. They have the ability to secrete proteins with high efficiency into the growth medium, which allows cost-effective downstream purification processing. Some of the most interesting and challenging heterologous proteins are recombinant antibodies and antibody fragments. They are important and suitable tools in medical research for analytics, diagnostics and therapy. The smallest conventional antibody fragment with high-affinity binding to an antigen is the single-chain fragment variable (scFv). Here, different strains of the genus Bacillus were investigated using diverse cultivation systems for their suitability to produce and secret a recombinant scFv.
  
  
  RESULTS
  Extracellular production of lysozyme-specific scFv D1.3 was realized by constructing a plasmid with a xylose-inducible promoter optimized for Bacillus megaterium and the D1.3scFv gene fused to the coding sequence of the LipA signal peptide from B. megaterium. Functional scFv was successfully secreted with B. megaterium MS941, Bacillus licheniformis MW3 and the three Bacillus subtilis strains 168, DB431 and WB800N differing in the number of produced proteases. Starting with shake flasks (150 mL), the bioprocess was scaled down to microtiter plates (1250 µL) as well as scaled up to laboratory-scale bioreactors (2 L). The highest extracellular concentration of D1.3 scFv (130 mg L-1) and highest space-time-yield (8 mg L-1h-1) were accomplished with B. subtilis WB800N, a strain deficient in eight proteases. These results were reproduced by the production and secretion of a recombinant penicillin G acylase (Pac).
  
  
  CONCLUSIONS
  The genus Bacillus provides high potential microbial host systems for the secretion of challenging heterologous proteins like antibody fragments and large proteins at high titers. In this study, the highest extracellular concentration and space-time-yield of a recombinant antibody fragment for a Gram-positive bacterium so far was achieved. The successful interspecies use of the here-designed plasmid originally optimized for B. megaterium was demonstrated by two examples, an antibody fragment and a penicillin G acylase in up to five different Bacillus strains.
authors.affiliation: Institute of Biochemical Engineering, Technische Universität Braunschweig, Rebenring 56, 38106, Braunschweig, Germany.|Center of Pharmaceutical Engineering (PVZ), Technische Universität Braunschweig, Franz-List-Straße 35a, 38106, Braunschweig, Germany.|Braunschweig Centre of Systems Biology (BRICS), Technische Universität Braunschweig, Rebenring 56, 38106, Braunschweig, Germany.
authors.forename: Antonia
authors.initials: A
authors.lastname: Lakowitz
authors.affiliation: Institute of Biochemical Engineering, Technische Universität Braunschweig, Rebenring 56, 38106, Braunschweig, Germany.|Center of Pharmaceutical Engineering (PVZ), Technische Universität Braunschweig, Franz-List-Straße 35a, 38106, Braunschweig, Germany.|Braunschweig Centre of Systems Biology (BRICS), Technische Universität Braunschweig, Rebenring 56, 38106, Braunschweig, Germany.
authors.forename: Rainer
authors.initials: R
authors.lastname: Krull
authors.affiliation: Braunschweig Centre of Systems Biology (BRICS), Technische Universität Braunschweig, Rebenring 56, 38106, Braunschweig, Germany. r.biedendieck@tu-braunschweig.de.|Institute of Microbiology, Technische Universität Braunschweig, Rebenring 56, 38106, Braunschweig, Germany. r.biedendieck@tu-braunschweig.de.
authors.forename: Rebekka
authors.initials: R
authors.lastname: Biedendieck
mesh_terms: D001407:Bacillus / Q000145:classification / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D001410:Bacillus megaterium / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics; D019149:Bioreactors; D003470:Culture Media; D007218:Industrial Microbiology / Q000379:methods; D010399:Penicillin Amidase / Q000235:genetics / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism; D010957:Plasmids; D011401:Promoter Regions, Genetic / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000276:immunology / Q000378:metabolism; D057127:Single-Chain Antibodies / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics* / Q000276:immunology
chemical_list: D001426:Bacterial Proteins; D003470:Culture Media; C079999:LipA protein, Bacteria; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; D010447:Peptide Hydrolases; D010399:Penicillin Amidase
keywords: Antibody fragment D1.3 scFv; Bacillus licheniformis; Bacillus megaterium; Bacillus subtilis; Proteases; Recombinant; Scale-down cultivation; Scale-up cultivation; Secretion