This project focused on designing peptide sequences that bind directly to unmodified magnetic nanoparticle (MNP) surfaces, serving as affinity tags for protein purification. This innovation eliminates the need for costly and degradable surface ligands typically used in affinity systems. The research involved identifying potential binders through peptide array experiments and examining their adsorption and desorption behaviors. Notably, negatively charged peptides exhibited the highest binding scores in Tris buffered saline but showed minimal binding in phosphate and citrate systems at the same pH. This phenomenon was attributed to the adsorption of anionic buffer molecules onto the particles, altering the available surface, as evidenced by zeta potential measurements. The buffer effect was utilized to desorb MNP from negatively charged peptide spots. Positive peptides bound strongly but irreversibly to MNP. Tags based on glutamate and a (RH)4-tag were fused to green fluorescent protein (GFP) via molecular cloning, with untagged and glycine-tagged variants serving as controls. The fusion proteins underwent chromatographic purification, revealing differences in binding behavior linked to tag exposure. A combination of freeze-thaw cycles and anion exchange chromatography emerged as an effective purification method. The binding behavior observed in peptide arrays was confirmed in solution, with E8-, E6-, and (RH)4-tagged proteins
