Characterization of Zinc-binding Heptapeptides as a Replacement for the His Tag in Improved Protein Purification Efficiency

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Chemistry

Date of Award

Summer 8-18-2025

Abstract

Zinc-binding peptides play a critical role in various biological and biochemical applications, particularly in metal coordination and protein purification. The ability of peptides to bind metal ions is essential for protein stability and affinity-based purification techniques such as immobilized metal affinity chromatography (IMAC). Traditional purification strategies often rely on polyhistidine (His Tag) sequences. However, alternative peptide motifs with enhanced metal-binding properties could improve efficiency and selectivity. In this research, the zinc-binding properties of novel acetylated heptapeptides, ac-X1-X2-Gly3-Pro4-Tyr5-X6-X7, where X1X2X6X7 = His1Cys2His6Cys7 (HCHC), Asp1Cys2His6Cys7 (DCHC) and Asp1Asp2His6Cys7 (DDHC) were investigated. The zinc-binding properties of the heptapeptides were studied through advanced techniques including Ultra Violet-Visible (UV-Vis) Spectroscopy, molecular modeling, pH-dependent affinity studies, and zinc chelating resin binding experiments. These techniques were used to assess whether the heptapeptides could serve as an effective alternative to the His Tag (7×His) for metal-affinity purification applications. Following the competitive Zn-binding studies, Zn(II)-chelating resin experiments were conducted on the heptapeptides to show their binding efficiencies using divalent zinc, coordinated by iminodiacetate, coupled to 6% cross-linked agarose beads, to mimic the IMAC process. The binding efficiencies of the heptapeptides were compared to the standard 7×His Tag using ion mobility – mass spectrometry with an internal standard method. Further analyses were conducted through UV-Vis spectroscopy to quantify the binding efficiency and selectivity of the peptides. The results indicated that the ZBP HCHC exhibited superior binding to the Zn(II)-resin compared to the 7×His Tag, maintaining strong retention under conditions typically used in IMAC purification. HCHC was not only able to bind Zn(II) more effectively than the His Tag but also demonstrated efficient elution from the resin under mild pH 3.9 conditions. This characteristic is particularly advantageous for affinity purification workflows, as it reduces the need for harsh elution conditions that can compromise protein integrity and yield. The improved performance of HCHC in both competitive binding, UV spectroscopy and resin-based assays highlights its potential as a superior alternative to the traditional His Tag in metal-affinity purification, offering enhanced binding efficiency, selectivity, and ease of recovery.

Advisor

Laurence Angel

Subject Categories

Chemistry | Physical Sciences and Mathematics

Link to Full Text

Share

COinS