Evaluating the Binding Efficiency of Alternative Histidine Affinity Tags to Zn and Ni Chelating Resins

Efficient binding of short 5–8 histidine (His) sequences to metal chelating resins is fundamental to modern recombinant protein purification. However, the homogeneous nature of the His repeat sometimes gives problems due to nonspecific binding of host proteins to the resin, the harsh elution conditions that can denature the recombinant protein, and structural interference with protein folding. This study investigates whether incorporating proline (Pro), tyrosine (Tyr), and cysteine (Cys) within these sequences, to produce a more heterogeneous tag, affects their ability to bind to chelating resins composed of divalent zinc or nickel ions chelated by iminodiacetate on cross‐linked agarose beads. Binding and elution efficiency from these resins was assessed using ion mobility–mass spectrometry, UV absorption spectroscopy, and high‐performance liquid chromatography, using pH 8 for binding and either mildly acidic pH 4–pH 5.5 or excess imidazole 0.1–0.5 M for elution. Overall, the findings indicate that short rationally designed His‐containing peptides can rival the performance of 7×His tags in immobilized metal affinity chromatography (IMAC) applications. By fine‐tuning the sequence composition, it is possible to optimize both binding affinity and elution efficiency for specific metal resins and experimental conditions indicating their potential as alternative affinity tags in purification workflows.