Background: Magnetic nanoparticles (MNPs) are becoming more important as carriers, because of their large specific surface area and easy separability. They are increasingly used in enzyme technology, diagnostics, and drug delivery. Major results: For the directed and almost irreversible immobilization of proteins on MNPs, we have developed a new selective (His-Arg) 4 peptide-tag, that binds fusion proteins directly from an E. coli cell lysate to non-functionalized, low-cost MNPs. Using the immobilization of an ene-reductase as an example, we could demonstrate that the fusion with this tag increases thermostability without reducing overall activity (ER w/o tag: t 1/2 = 3.7 h, (HR) 4-ER: t 1/2 = 9.9 h). Immobilization by adsorption in Tris buffer resulted in very high enzyme loads with approx. 380 mg g-1 and 67% residual activity. The immobilization on the MNPs allowed a fast concentration, buffer exchange, and reuse. While about 50% of the activity was lost after the first reuse, we were able to show that the activity did not decrease further and was stable for another nine cycles. Conclusion: According to our studies, our tag highly works for any kind of immobilization on MNPs and holds the potential for enzyme immobilizations as well as for drug delivery and sensors. K E Y W O R D S affinity tag, ene-reductase, enzyme immobilization, iron oxides, magnetic nanoparticles 1 INTRODUCTION Magnetic nanoparticles are used in all kinds of applications, such as enzyme immobilization, bioimaging, biosensors, drug delivery, enrichment of bacteria, and many more. [1-5] The high specific surface areas Abbreviations: (HR) 4 , dipeptide histidine-arginine repeated four times; AEX, anion exchange chromatography; Arg, arginine; BCA, bicinchoninic acid; ER, ene-reductase; ER w/o tag, ene-reductase without a tag; FMN, flavin mononucleotide; GFP, green fluorescent protein; His, histidine; HPLC, high-performance liquid chromatography; IMAC, immobilized metal affinity chromatography; Lys, lysine; MNPB, bound proteins on magnetic nanoparticles; MNPs, magnetic nanoparticles; MNPS, supernatant of magnetic nanoparticles after magnetic separation; MNPW, wash supernatant of magnetic nanoparticles after magnetic separation; NADH, nicotinamide adenine dinucleotide; NADPH, nicotinamide adenine dinucleotide phosphate; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SEC, size exclusion chromatography; TBS, Tris-buffered saline This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

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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