Development a Magnetic Nanoparticle-Assisted Biotinylation System: Synthesis, Purification and Activity Assessment of MagR-fused TurboID Biotin Ligase Enzyme

General Information and Purpose: Enzymatic biotinylation is widely used for protein labelling, but free enzyme contamination may limit its efficiency and specificity in some condition. In this study, TurboID was fused with the Magnetoreceptor protein (MagR) to create a recombinant fusion enzyme. Its magnetic nanoparticle binding and biotinylation activity were evaluated. The aim was to develop a magnetically controllable, low-contamination system and broaden the applications of enzymatic biotinylation Materials and Methods. Gene sequences were sourced from NCBI GenBank and designed with flexible linkers to allow proper fusion. These were cloned into expression plasmids and introduced into E. coli for protein production. After purification, enzyme activity was assessed, and the proteins were then attached to magnetic nanoparticles (Fe3O4-SiO2). Finally, their biotinylation activity was tested in cell culture Findings: TurboID-MagR enzyme production was carried out with an MBP-tag. The enzyme was successfully purified, activity asses performed by comparing TurboID and bound to magnetic nanoparticles as designed, and cell surface biotinylation activity was successfully demonstrated in cell culture experiments. Discussion: In this study, the enzyme was successfully produced, although a slight decrease in activity and aggregation was observed during purification. Despite these challenges, the system worked successfully in vitro settings, promising for broader use in biological applications. Conclusion: TurboID-MagR system was successfully produced and remained active despite some limitations. Its magnetic controllability and activity highlight its potential for broader, contamination-reduced applications.