View - ResearchGate

9
Preparation, Analysis and Use of an Affinity Adsorbent
for the Purification of GST Fusion Protein
Gareth M. Forde
Summary
Methods are presented for the preparation, ligand density analysis and use of an affinity
adsorbent for the purification of a glutathione S-transferase (GST) fusion protein in packed
and expanded bed chromatographic processes. The protein is composed of GST fused to a
zinc finger transcription factor (ZnF). Glutathione, the affinity ligand for GST purification,
is covalently immobilized to a solid-phase adsorbent (Streamline ). The GST–ZnF fusion
protein displays a dissociation constant of 0.6 × 10 −6 M to glutathione immobilized
to Streamline . Ligand density optimization, fusion protein elution conditions (pH and
glutathione concentration) and ligand orientation are briefly discussed.
Key Words: Key Words: GST fusion protein; affinity purification; chromatography;
expanded bed adsorption.
1. Introduction
Purification based on targeted affinity interactions offers high selectivity
and facile purification of biomolecules including the capture of products from
complex feed stocks (1,2,3). The use of affinity ligands leads to an increased
adsorbent selectivity, resulting in higher degrees of purification and potentially
higher capacities of adsorbent for the target. Due to its high selectivity, affinity
chromatography is a preferred tool in the downstream processing of high-value
biomolecules of therapeutic interest (4).
From: Methods in Molecular Biology, vol. 421: Affinity Chromatography: Methods and Protocols, Second Edition
Edited by: M. Zachariou © Humana Press, Totowa, NJ
125