Superparamagnetic nanoparticles have recently gained much attention due to their broad range of applicability including medical in vivo technologies, sensors, and as supports for catalysts. As magnetic affinity materials, they can be utilized for the development of new purification strategies for pharmaceuticals and other target molecules from crude lysates. Here, a short peptide tag based on a glutamate sequence is introduced and the adsorption of pure protein as well as protein from crude cell lysate at different conditions is demonstrated. Fused to a model protein this tag can be used to recognize and purify this protein from a fermentation broth by bare iron oxide nanoparticles (BIONs). Binding of up to 0.2 g protein per g nanoparticles can be achieved and recovered easily by switching to a citrate buffered system. For a deeper understanding of the separation process, the aggregation and agglomeration of the nanoparticle protein systems were monitored for binding and elution steps. Furthermore, an upscaling of the process to the liter scale and the separation of a green fluorescent protein (GFP) containing the affinity tag to purities of 70% from Escherichia coli fermentation broth was possible in a one step process by means of high gradient magnetic separation (HGMS).

Magnetic separation is a promising alternative to conventional methods in downstream processing. This can facilitate easier handling, fewer processing steps, and more sustainable processes. Target materials can be extracted directly from crude cell lysates in a single step by magnetic nanoadsorbents with high-gradient magnetic fishing (HGMF). Additionally, the use of hazardous consumables for reducing downstream processing steps can be avoided. Here, we present proof of principle of one-step magnetic fishing from crude Escherichia coli cell lysate of a green fluorescent protein (GFP) with an attached hexahistidine (His 6 )-tag, which is used as the model target molecule. The focus of this investigation is the upscale to a liter scale magnetic fishing process in which a purity of 91% GFP can be achieved in a single purification step from cleared cell lysate. The binding through the His 6 -tag can be demonstrated, since no significant binding of nontagged GFP toward bare iron oxide nanoparticles (BIONs) can be observed. Nonfunctionalized BIONs with primary particle diameters of around 12 nm, as used in the process, can be produced with a simple and low-cost coprecipitation synthesis. Thus, HGMF with BIONs might pave the way for a new and greener era of downstream processing.

Magnetic nanoparticles (MNP) are intensively investigated for applications in nanomedicine, catalysis and biotechnology, where their interaction with peptides and proteins plays an important role. However, the characterisation of the interaction of individual amino acids with MNP remains challenging. Here, we classify the affinity of 20 amino acid homo-hexamers to unmodified iron oxide nanoparticles using peptide arrays in a variety of conditions as a basis to identify and rationally design selectively binding peptides. The choice of buffer system is shown to strongly influence the availability of peptide binding sites on the MNP surface. We find that under certain buffer conditions peptides of different charges can bind the MNP and that the relative strength of the interactions can be modulated by changing the buffer. We further present a model for the competition between the buffer and the MNP's electrostatically binding to the adsorption sites. Thereby, we demonstrate that the charge distribution on the surface can be used to correlate the binding of positively and negatively charged peptides to the MNP. This analysis enables us to engineer the binding of MNP on peptides and contribute to better understand the bio-nano interactions, a step towards the design of affinity tags for advanced biomaterials.Magnetic nanoparticles (MNP) are widely used for the purification of nucleic acids and other biological molecules [1][2][3][4] . MNP are also employed in the immobilisation of enzymes 5,6 , for biomedical applications such as drug delivery and hyperthermia in cancer treatment and as contrast agents for magnetic resonance imaging 7,8 . Their superparamagnetic behaviour allows for their manipulation by an external magnetic field to easily accumulate MNP in a desired area 9 . In addition, MNP have also spiked interest in the field of catalytic reaction engineering 10 . For most applications, MNP have to be functionalised to allow for a selective binding of the target molecule. This is presently achieved by attaching metal-ion chelating molecules, e.g. nitrilotriacetic acid, to the MNP surface, which then bind His-tagged biomolecules 11,12 . Drawbacks of this method are the leakage of toxic metal ions and instability of the surface functionalisation 13,14 . Alternative surface modifications for protein adsorption on magnetic particles are glutathione, streptavidin, biotin or protein A, all of which lead to high costs running in the thousands of Euros per gram 15 . Use of bare superparamagnetic iron oxides thus offers decisive advantages for industrial applications mainly due to the easy, rapid and low-cost synthesis and the absence of degradable functional surface groups. We undertake here the first systematic study of peptide-MNP interactions of bare MNP to ultimately develop peptides that can be genetically engineered into proteins as tags and strongly bind to the nonfunctionalised MNP. The key to the design of high-affinity peptide tags lies in an in-depth understanding of surface-peptide recognition patterns 16 ....

Magnetic metal oxide nanoparticles demonstrate great applicability in several fields such as biotechnology, medicine and catalysis. A stable, magnetic and low-cost material, nanoscale magnetite, is an interesting adsorbent for protein purification. Downstream processing can account for up to 80% of the total production costs in biotechnological production. As such, the development of new innovative separation methods can be regarded as highly profitable. While short peptide sequences can be used as specific affinity tags for functionalised adsorber surfaces, they need expensive affinity ligands on the particle surface for adsorption. In order to identify peptide tags for several non-functionalised inorganic surfaces, different binding conditions to iron oxide nanoparticles are evaluated. Therefore, magnetite nanoparticles in a range of 5-20 nm were synthesised with a co-precipitation method. Zeta potential measurements indicated an amphiphilic surface with an isoelectric point in the neutral pH region. Glutamic acid-based homo-peptides were used as affinity peptides for the magnetite nanoparticles. We demonstrate a dependence of the binding affinity of the peptides on pH and buffer ions in two different experimental set-ups. The nature of surface coordination for glutamic acid-based peptides can be demonstrated with different spectroscopic approaches such as infrared spectroscopy (IR), Raman spectroscopy and circular dichroism spectroscopy (CD). We want to emphasise the importance of physicochemical properties such as surface energy, polarity, morphology and charge. These parameters, which are dependent on the environmental conditions, play a crucial role in peptide interactions with iron oxide surfaces. The understanding of the adsorption of simple biomolecules on nanoscale metal oxide surfaces also represents the key to the even more complex interactions of proteins at the bio-nano interface. From the identification of interaction patterns and an understanding of the adsorption of these peptides, the up-scaling to tagged model proteins facilitates the possibility of an industrial magnetic separation process and might therefore reduce time and costs in purification processes.

No abstract