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126 Forde
Utilization of the GST-glutathione affinity interaction in a chromatographic
process has a number of distinct advantages. It enables a straightforward
detection protocol via the use of an enzyme activity assay, a reproducible
purification strategy from lysed cell culture via adsorption to immobilized
glutathione, high selectivity and a convenient strategy for the regeneration
of the affinity adsorbent. An enzyme activity assay facilitates the fast, highthroughput
assaying of fractions for the quantitative measurement of GST
protein concentration.
Presented is a process for the purification of a GST fusion protein. The
protein is composed of GST fused to a zinc finger transcription factor (ZnF).
The bi-functional fusion protein displays dual affinity for glutathione, via the
GST segment, and a specific DNA sequence, via the zinc-finger motif. The
protein was ultimately designed for the affinity purification of plasmid DNA.
The zinc finger is also known as the Cys 2 His 2 zinc finger and is a transcription
factor that regulates the expression of proteins by binding specifically to certain
DNA sequences. The production of a zinc finger protein that displayed affinity
for a 9-base pair sequence was first reported by Desjarlais and Berg (5).
In this work, glutathione is covalently immobilized to a solid-phase adsorbent
(Streamline ). The primary biological function of glutathione is to act as a
non-enzymatic reducing agent to help keep cysteine thiol side chains in a
reduced state on the surface of proteins, which has led to its use as a medicinal
antioxidant. Glutathione prevents oxidative stress in most cells and helps to trap
free radicals that can damage DNA and RNA. GST catalyzes the nucleophilic
attack of the sulphur atom of the glutathione on electrophilic groups of a variety
of hydrophobic substrates, including herbicides, insecticides and carcinogens
(6,7). The GST–ZnF fusion protein displayed a dissociation constant of 0.6 ×
10 −6 M to glutathione immobilized to Streamline , which is similar to that
reported for recombinant GST binding to a glutathione-Sepharose affinity
adsorbent of 1.15 × 10 −6 M (8).
Packed bed and expanded bed operation modes were employed to purify the
target GST fusion protein. Expanded bed adsorption (EBA) is a quasi-packed
bed unit operation through which large particulates (such as suspended solids
in non-clarified feeds) can pass. EBA enables bio-target recovery directly from
particulate containing feedstocks like cell homogenates or fermentation broth
(for extracellular bio-targets). EBA can complete the functions of clarification,
concentration and purification in one stage and thereby increase the total yield
and reduce the operation time of a process system by reducing the number
of stages.