Author ORCID Identifier

0009-0001-9962-4643

Document Type

Thesis

Date of Award

Spring 5-1-2026

Keywords

tag-free protein purification, self-cleaving tag, hedgehog proteins, HAC8

Degree Name

Biochemistry (BS)

Department

CHEMISTRY

First Advisor

Brian Callahan

Second Advisor

Christof Grewer

Third Advisor

Minfei Su

Abstract

Because removing extraneous purification tag sequences from native proteins can be challenging and costly, there is demand for simple all-in-one techniques that directly furnish a tagless, functional protein-of-interest (POI). Current tagless purification systems are based on intein biochemistry; however, intein-based purification requires expensive, specialized resins and the POI often must be fused to the intein’s C-terminus. In this thesis, I present a novel approach for tagless POI purification in which the Drosophila melanogaster hedgehog protein catalytic domain (HhC) is repurposed as a small-molecule inducible, self-cleaving element dubbed Gtag. With Gtag, the POI is fused to the N-terminus of HhC/Gtag with an intervening glycine residue, POI-Gly-Gtag. I have cloned several POI-Gly-Gtag-His6 constructs and expressed the resulting precursor proteins in E. coli. The Gtag purification scheme I developed involves three steps: (i) immobilize recombinant POI-Gly-Gtag His6 protein on standard Ni-NTA resin, (ii) wash away loosely bound contaminate protein (iii) induce POI-Gly-↓-Gtag-His6 endoproteolysis by adding noncovalent hydrolysis-activating compounds (HAC8) and collect the resulting elution. The HAC8 compound used here to induce Gtag endoproteolysis was identified in an earlier unrelated study by the Callahan group. Using this Gtag approach, I demonstrate the successful purification of diverse POIs in tag-free, functional form, including: Nanoluciferase, mCherry, T4 lysozyme, GFP nanobody, and the human Sonic hedgehog signaling protein.

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