Integrated in situ -purification of recombinant proteins from Bacillus megaterium cultivation using SPION in stirred tank reactors

Gädke, Johannes; Thies, J.-W.; Kleinfeldt, Lennart; Kalinin, Artem; Starke, Gereon; Lakowitz, Antonia; Biedendieck, Rebekka; Garnweitner, Georg; Dietzel, Andreas; Krull, Rainer

doi:10.1016/j.bej.2017.07.001

Cited by 8 publications

(9 citation statements)

References 25 publications

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“…The magnetic separability of selected aggregates with different iron oxide-to-silica compositions was evaluated using the basic reactor setup presented by Gädke et al 42 For the basic separation efficiency experiments, an abiotic system was used, suspending the nonfunctionalized particles in deionized water. The complete reactor volume was cycled through the external loop seven times to ensure comparability with the previous findings.…”

Section: Results and Discussionmentioning

confidence: 99%

“…The integrated automation of a lab-scale stirred-tank reactor for the production, in situ separation, and purification of recombinant proteins utilizing the functionalized spray-dried pure SPION aggregates was reported by Gädke et al 42…”

Section: Results and Discussionmentioning

confidence: 99%

“…The in situ separation of the recombinant protein (concentration reached, 90 mg mL –1 ) from the growth medium occurred during the exponential growth phase when functionalized particles were added to a final concentration of 0.6 mg mL –1 and magnetically separated after 10 min by pumping the medium through an external loop with an attached permanent magnet. 42 The application concept is depicted in Figure 1B. Subsequently, the loaded particles were washed, and recombinant proteins were eluted from the particles with appropriate buffers (compare Tables S2 and S3) before the particles were regenerated and collected to be reloaded with Ni 2+ (see Tables S4 and S5) to regain the specific absorption capacity.…”

Section: Experimental Sectionmentioning

confidence: 99%

“…The magnetic separability of selected SD aggregates was tested using aqueous suspensions in a laboratory setup of a circulation loop with handheld magnets as described elsewhere. 42…”

Section: Experimental Sectionmentioning

confidence: 99%

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Spray-Dried Hierarchical Aggregates of Iron Oxide Nanoparticles and Their Functionalization for Downstream Processing in Biotechnology

et al. 2019

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In this work, the structuring of iron oxide nanoparticles via spray-drying (SD) of aqueous suspensions is investigated, leading to micrometer-sized aggregates with saturation magnetization comparable to that of the individual nanoparticles. Interestingly, the superparamagnetic behavior is retained despite the multicore structure. Modification of the aggregates via the addition of silica nanoparticles to the suspension allows for control of the resulting magnetization by adjusting the iron oxide content. Moreover, the morphology of the produced aggregates is gradually shifted from irregular inflated-like shapes in case of pure iron oxide aggregates to reach spherical structures when bringing the silica content to only 20%. The aggregates with different magnetization can be effectively separated in a simple column with an attached permanent magnet. Functionalization of pure iron oxide aggregates with a previously coupled ligand holding a nitrilotriacetic acid (NTA)-like moiety and subsequent loading with Ni2+ ions leads to the ability to bind 6-histidine (His6)-tagged target proteins via chelation complexes for magnetic separation. The application of the presented system for the purification of recombinant protein A in multiple cycles is shown. The recyclability of the separation system in combination with the high degree of magnetic separation is promising for future applications in the field of preparative in situ protein purification.

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Section: Results and Discussionmentioning

confidence: 99%

Section: Results and Discussionmentioning

confidence: 99%

Section: Experimental Sectionmentioning

confidence: 99%

“…The magnetic separability of selected SD aggregates was tested using aqueous suspensions in a laboratory setup of a circulation loop with handheld magnets as described elsewhere. 42…”

Section: Experimental Sectionmentioning

confidence: 99%

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Spray-Dried Hierarchical Aggregates of Iron Oxide Nanoparticles and Their Functionalization for Downstream Processing in Biotechnology

et al. 2019

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“…When combined with extracellular protein production, affinity tags provide rapid tools for protein purification, especially along with continuous cultivation processes (Gädke et al 2017a;Gädke et al 2017b). Moreover, a codon plus system employing the co-expression of genes for tRNAs with rare codons often found in heterologous target genes showed a general positive effect on recombinant protein production by P. megaterium (Finger et al 2015) (Fig.…”

Section: Secretion Of Recombinant Proteins With P Megateriummentioning

confidence: 99%

The “beauty in the beast”—the multiple uses of Priestia megaterium in biotechnology

Biedendieck

Knuuti

Jahn

2021

Appl Microbiol Biotechnol

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Over 30 years, the Gram-positive bacterium Priestia megaterium (previously known as Bacillus megaterium) was systematically developed for biotechnological applications ranging from the production of small molecules like vitamin B12, over polymers like polyhydroxybutyrate (PHB) up to the in vivo and in vitro synthesis of multiple proteins and finally whole-cell applications. Here we describe the use of the natural vitamin B12 (cobalamin) producer P. megaterium for the elucidation of the biosynthetic pathway and the subsequent systematic knowledge-based development for production purposes. The formation of PHB, a natural product of P. megaterium and potential petro-plastic substitute, is covered and discussed. Further important biotechnological characteristics of P. megaterium for recombinant protein production including high protein secretion capacity and simple cultivation on value-added carbon sources are outlined. This includes the advanced system with almost 30 commercially available expression vectors for the intracellular and extracellular production of recombinant proteins at the g/L scale. We also revealed a novel P. megaterium transcription-translation system as a complementary and versatile biotechnological tool kit. As an impressive biotechnology application, the formation of various cytochrome P450 is also critically highlighted. Finally, whole cellular applications in plant protection are completing the overall picture of P. megaterium as a versatile giant cell factory. Key points • The use of Priestia megaterium for the biosynthesis of small molecules and recombinant proteins through to whole-cell applications is reviewed. • P. megaterium can act as a promising alternative host in biotechnological production processes.

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Magnetic Nanoparticles for Protein Separation and Purification

Vedarethinam,

Jeevanandam,

Acquah

et al. 2023

Methods in Molecular Biology

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Integrated in situ -purification of recombinant proteins from Bacillus megaterium cultivation using SPION in stirred tank reactors

Cited by 8 publications

References 25 publications

Spray-Dried Hierarchical Aggregates of Iron Oxide Nanoparticles and Their Functionalization for Downstream Processing in Biotechnology

Spray-Dried Hierarchical Aggregates of Iron Oxide Nanoparticles and Their Functionalization for Downstream Processing in Biotechnology

The “beauty in the beast”—the multiple uses of Priestia megaterium in biotechnology

Magnetic Nanoparticles for Protein Separation and Purification

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