Conjugating his-tagged proteins to magnetic nanoparticles: tips and challenges

Resumen del póster presentado al 5th Multistep Enzyme Catalyzed Processes Congress (MECPC), celebrado online del 13 al 16 de septiembre de 2021.The histidine tag (His-tag) is one of the most used affinity-tag for protein purification due to its small size and versatility. Agarose and sepharose beads containing nitriloacetic acid (NTA) transition metal derivatives are widely used for the purification of His-tagged proteins, thanks to their high affinity to the His-tag genetically fused to the protein of interest [1]. The same chemistry can be used to conjugate enzymes to magnetic nanoparticles (MNPs) with the aim of tuning their activity by magnetic heating [2, 3]. Within the frame of the FET-OPEN project HOTZYMES (https://www.hotzymes.eu), different MNPs have been synthetized and coated with polyacrylic acid and dimercaptosuccinic acid, and then were further functionalized with NTA-Cu2+ as His-tag chelating agent. Different proteins were expressed as His-tag variants and immobilized on the MNPs, including monomeric (superfolded GFP), dimeric (C. violaceum transaminase, CvTA; C. uda cellobiose phosphorylase, CuCbP), and tetrameric (B. stearothermophilus alcohol dehydrogenase, ADH) variants. While for the monomeric protein selected as model no difficulties in the bioconjugation processes were observed, when using dimeric or tetrameric enzymes the aggregation of the MNPs occurs very easily due to crosslinking between the nanoparticles. This colloidal destabilization of the MNPs is favored due to its high surface area and the presence of several tags per enzyme molecule. To avoid this situation, different strategies have been developed: saturation of the binding site of the nanoparticles, presence of a small percentage of imidazole in reaction, changes in the incubation conditions (pH, ionic strength, …). First positive results confirm that by playing with different factors it is possible to conjugate different His-tagged enzymes to very different MNPs in terms of size, shape, surface area, and colloidal stability. Actually, it is possible to avoid protein and MNPs aggregation while obtaining good activity yields for the conjugated enzymes and maintaining the magnetic heating capacity of the MNPs.The research for this work has received funding from the European Union (EU) project HOTZYMES (grant agreement n° 829162) under EU’s Horizon 2020 Programme Research and Innovation actions H2020-FETOPEN-2018-2019-2020-01. Authors also thank Spanish MINECO project BIO2017-84246-C2-1-R, DGA and Fondos Feder (Bionanosurf E15_17R).Peer reviewe