Investigation of Novel Peptide Tags for Zinc and Nickel Binding: Alternatives to Traditional His-tag Protein Purification Systems
Document Type
Thesis
Degree Name
Master of Science (MS)
Department
Chemistry
Date of Award
9-1-2026
Abstract
The recombinant protein purification process is the most common approach for purifying proteins after expression in mammalian cells. In this process, the His-tag, which consists of 6–10 histidine residues, is the traditional affinity tag served in immobilized metal affinity chromatography (IMAC) because it binds well to metals such as zinc and nickel. Despite His-tags enabling efficient affinity purification, their limitations include nonspecific binding, metal ion interference, potential effects on protein structure and function, and the need for additional purification or tag-removal steps. In this study, four heptapeptides that follow the general formula Ac-H₁-Aa₂-Gly₃-Pro₄-Aa₅-His₆-Aa₇ (where Aa₂ = His₂ or Cys₂, Aa₅ = Tyr₅ or Gly₅, and Aa₇ = His₇ or Cys₇) were investigated and compared with the 7×His tag. The affinity of the proposed heptapeptides was studied using two different approaches. Competition analysis between two different sets of heptapeptides and zinc ions over a pH range of 4 to 9 was performed using ESI-MS. Binding and elution percentages with zinc and nickel resins were evaluated using ESI-MS and UV-Vis spectroscopy to compare the proposed tags with the His-tag. The study revealed that HCYHH exhibited the highest binding among 7×His and HHGHH in competition analysis over a pH range of 4–9, whereas HHYHC exhibited the highest binding in the other set among HHYHH and 7×His. This suggests that HCYHH and HHYHC have strong affinity for zinc and nickel resins. In resin analysis, HHYHH showed high binding affinity for both zinc and nickel resins, with superior elution percentages under mild conditions (acetate, pH 4.0). HCYHH and HHYHC showed high binding affinity only to the zinc resin, with superior elution percentages compared with 7×His. HHGHH showed poor binding affinity to both resins. These results indicate that HHYHH, HCYHH, and HHYHC are promising alternatives to the ubiquitous 7×His tag, offering slightly lower binding but significantly higher recovery.
Advisor
Laurence Angel
Subject Categories
Chemistry | Physical Sciences and Mathematics
Recommended Citation
Arar, Mohammad Raid, "Investigation of Novel Peptide Tags for Zinc and Nickel Binding: Alternatives to Traditional His-tag Protein Purification Systems" (2026). Electronic Theses & Dissertations. 1388.
https://lair.etamu.edu/etd/1388
