title: In situ affinity purification of his-tagged protein A from Bacillus megaterium cultivation using recyclable superparamagnetic iron oxide nanoparticles.
abstract: This paper discusses the use of recyclable functionalized nanoparticles for an improved downstream processing of recombinant products. The Gram-positive bacterium Bacillus megaterium was used to secrete recombinant protein A fused to a histidine tag into the culture supernatant in shaker flasks. Superparamagnetic iron oxide nanoparticles functionalized with 3-glycidoxypropyl-trimethoxysilane-coupled-nitrilotriacetic-acid groups (GNTA-SPION) were synthesized and added directly to the growing culture. After 10min incubation time, >85% of the product was adsorbed onto the particles. The particles were magnetically separated using handheld neodymium magnets and the product was eluted. The GNTA-SPION were successfully regenerated and reused in five consecutive cycles. In the one-step purification, the purity of the product reached >99.9% regarding protein A. A very low particle concentration of 0.5g/L was sufficient for effective product separation. Bacterial growth was not influenced negatively by this concentration. Particle analysis showed similar properties between freshly synthesized and regenerated GNTA-SPION. The overall process efficiency was however influenced by partial disintegration of particle agglomerates and thus loss of particles. The demonstration of very fast in situ product removal from growing bacterial culture combined with a very high product purity within one step shows possibilities for automated large scale purification combined with recycling of biomass.
authors.affiliation: Institute of Biochemical Engineering, TU Braunschweig, Rebenring 56, 38106 Braunschweig, Germany; Center of Pharmaceutical Engineering (PVZ), TU Braunschweig, Germany; Braunschweig Integrated Centre of Systems Biology (BRICS), TU Braunschweig, Rebenring 56, 38106 Braunschweig, Germany.
authors.forename: Johannes
authors.initials: J
authors.lastname: Gädke
authors.affiliation: Center of Pharmaceutical Engineering (PVZ), TU Braunschweig, Germany; Institute for Particle Technology, TU Braunschweig, Volkmaroder Str. 5, 38104 Braunschweig, Germany.
authors.forename: Lennart
authors.initials: L
authors.lastname: Kleinfeldt
authors.affiliation: Institute of Biochemical Engineering, TU Braunschweig, Rebenring 56, 38106 Braunschweig, Germany; Center of Pharmaceutical Engineering (PVZ), TU Braunschweig, Germany; Braunschweig Integrated Centre of Systems Biology (BRICS), TU Braunschweig, Rebenring 56, 38106 Braunschweig, Germany.
authors.forename: Chris
authors.initials: C
authors.lastname: Schubert
authors.affiliation: Helmholtz Centre for Infection Research (HZI), Central Facility for Imaging, Inhoffenstr. 7, 38124 Braunschweig, Germany.
authors.forename: Manfred
authors.initials: M
authors.lastname: Rohde
authors.affiliation: Braunschweig Integrated Centre of Systems Biology (BRICS), TU Braunschweig, Rebenring 56, 38106 Braunschweig, Germany; Institute of Microbiology, TU Braunschweig, Spielmannstr. 7, 38106 Braunschweig, Germany.
authors.forename: Rebekka
authors.initials: R
authors.lastname: Biedendieck
authors.affiliation: Center of Pharmaceutical Engineering (PVZ), TU Braunschweig, Germany; Institute for Particle Technology, TU Braunschweig, Volkmaroder Str. 5, 38104 Braunschweig, Germany.
authors.forename: Georg
authors.initials: G
authors.lastname: Garnweitner
authors.affiliation: Institute of Biochemical Engineering, TU Braunschweig, Rebenring 56, 38106 Braunschweig, Germany; Center of Pharmaceutical Engineering (PVZ), TU Braunschweig, Germany; Braunschweig Integrated Centre of Systems Biology (BRICS), TU Braunschweig, Rebenring 56, 38106 Braunschweig, Germany. Electronic address: r.krull@tu-braunschweig.de.
authors.forename: Rainer
authors.initials: R
authors.lastname: Krull
mesh_terms: D065966:Absorption, Physicochemical; D001410:Bacillus megaterium / Q000737:chemistry* / Q000378:metabolism; D020099:Coated Materials, Biocompatible / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay; D006639:Histidine; D008280:Magnetics / Q000379:methods*; D058185:Magnetite Nanoparticles / Q000737:chemistry*; D008855:Microscopy, Electron, Scanning; D010316:Particle Size; D011994:Recombinant Proteins / Q000737:chemistry; D013205:Staphylococcal Protein A / Q000737:chemistry / Q000302:isolation & purification*; D013818:Thermogravimetry
chemical_list: D020099:Coated Materials, Biocompatible; D058185:Magnetite Nanoparticles; D011994:Recombinant Proteins; D013205:Staphylococcal Protein A; D006639:Histidine
keywords: Bacillus megaterium; In situ purification; Magnetic separation; Particle regeneration; Protein purification; SPION