Schwaminger Sebastian P, Fraga-García Paula, Blank-Shim Silvia A, Straub Tamara, Haslbeck Martin, Muraca Francesco, Dawson Kenneth A, Berensmeier Sonja
Bioseparation Engineering Group, Department of Mechanical Engineering and Department of Chemistry, Technical University of Munich, Garching 85748, Germany.
Centre for BioNano Interactions, School of Chemistry and Chemical Biology and Conway Institute for Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin D14 YH57, Ireland.
ACS Omega. 2019 Feb 21;4(2):3790-3799. doi: 10.1021/acsomega.8b03348. eCollection 2019 Feb 28.
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 cell lysate of a green fluorescent protein (GFP) with an attached hexahistidine (His)-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-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.
在下游加工中，磁分离是一种很有前景的传统方法替代方案。这可以使操作更简便、加工步骤更少且工艺更具可持续性。通过具有高梯度磁捕集（HGMF）的磁性纳米吸附剂，目标材料可以在单个步骤中直接从粗细胞裂解物中提取出来。此外，还可以避免使用有害消耗品来减少下游加工步骤。在此，我们展示了从带有六聚组氨酸（His）标签的绿色荧光蛋白（GFP）的粗细胞裂解物中进行一步磁捕集的原理证明，该GFP用作模型目标分子。本研究的重点是扩大到升规模的磁捕集过程，其中从澄清的细胞裂解物中通过单个纯化步骤可实现91%的GFP纯度。通过His标签的结合可以得到证明，因为未标记的GFP与裸氧化铁纳米颗粒（BIONs）之间未观察到明显的结合。该过程中使用的初级粒径约为12 nm的未功能化BIONs可以通过简单且低成本的共沉淀合成法制备。因此，使用BIONs的HGMF可能为下游加工的一个新的绿色时代铺平道路。
