In situ affinity purification of his-tagged protein A from Bacillus megaterium cultivation using recyclable superparamagnetic iron oxide nanoparticles

Gädke, Johannes; Kleinfeldt, Lennart; Schubert, Chris; Rohde, Manfred; Biedendieck, Rebekka; Garnweitner, Georg; Krull, Rainer

doi:10.1016/j.jbiotec.2016.11.018

Cited by 24 publications

(23 citation statements)

References 35 publications

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“…When GNTA-SPIONs are applied in the in situ purification in shaking flasks, an average particle loss of approximately 30% per cycle over five consecutive cycles is detected. 17 In contrast, on average, 93% of the GNTA-SD-SPIONs are retained during the separation process in shaking flasks.…”

Section: Results and Discussionmentioning

confidence: 99%

“…Eluted proteins were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) as described previously. 17 Finally, after further cultivation, the particles were added back into the bioreactor for an additional separation cycle.…”

Section: Experimental Sectionmentioning

confidence: 99%

“…Various approaches using the adsorption onto magnetic nanoparticles and their subsequent separation have been reported. 15−17 The magnetic separation can be improved by increasing the magnetization of the individual particles, which is achieved by combining multiple nanoparticles in a multicore particle with a bigger size. Usually this is done by embedding SPIONs in a polymer matrix.…”

Section: Introductionmentioning

confidence: 99%

“…While our previous study 17 was focused on the integration of the in situ purification of proteins from microbial cultivation using agglomerated functionalized nanoparticles, here we focus on the tailoring of the magnetic particles to improve separability and retention over consecutive cycles.…”

Section: Introductionmentioning

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: Experimental Sectionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

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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“…The use of MNPs in purifying his-tagged proteins is advantageous in improving adsorption capacity, simplifying operating procedures, and reducing time consuming. 27,28 MNPs chelating with metal ions enabled one-step immobilization and purication of his-tagged proteins. 29 Proteins of enzymes immobilized on MNPs are easily separated from cell lysates or reacting mixtures under the function of external magnetic force.…”

Section: Introductionmentioning

confidence: 99%

Synthesis of magnetic nanoparticles with an IDA or TED modified surface for purification and immobilization of poly-histidine tagged proteins

et al. 2020

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Direct capture and selective elution of a secreted polyglutamate‐tagged nanobody using bare magnetic nanoparticles

Zanker

Stargardt²,

Kurzbach

et al. 2022

Biotechnology Journal

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Background The secretion and direct capture of proteins from the extracellular medium is a promising approach for purification, thus enabling integrated bioprocesses. Major Results We demonstrate the secretion of a nanobody (VHH) to the extracellular medium (EM) and its direct capture by bare, non‐functionalized magnetic nanoparticles (MNPs). An ompA signal peptide for periplasmic localization, a polyglutamate‐tag (E8) for selective MNP binding, and a factor Xa protease cleavage site were fused N‐terminally to the nanobody. The extracellular production of the E8‐VHH (36 mg L–1) was enabled using a growth‐decoupled Escherichia coli‐based expression system. The direct binding of E8‐VHH to the bare magnetic nanoparticles was possible and could be drastically improved up to a yield of 88% by adding polyethylene glycol (PEG). The selectivity of the polyglutamate‐tag enabled a selective elution of the E8‐VHH from the bare MNPs while raising the concentration factor (5x) and purification factor (4x) significantly. Conclusion Our studies clearly show that the unique combination of a growth‐decoupled E. coli secretion system, the polyglutamate affinity tag, non‐functionalized magnetic nanoparticles, and affinity magnetic precipitation is an innovative and novel way to capture and concentrate nanobodies.

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In situ affinity purification of his-tagged protein A from Bacillus megaterium cultivation using recyclable superparamagnetic iron oxide nanoparticles

Cited by 24 publications

References 35 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

Synthesis of magnetic nanoparticles with an IDA or TED modified surface for purification and immobilization of poly-histidine tagged proteins

Direct capture and selective elution of a secreted polyglutamate‐tagged nanobody using bare magnetic nanoparticles

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