Bio-Rad Profinia™ Protein Purification Instrument - Manual

Automated Purification of a GST-Tagged Protein With the
Profinia
™ Protein Purification System: Comparison to Manual
Protein Purification Using Commercial Kits
fractions allows determination of the effectiveness of
purification. Automated desalting of the purified protein by
size exclusion chromatography is also possible without the
need for user intervention between the affinity and desalting
chromatography steps. When used with Profinia software, the
system displays purification data in real time and allows
generation and printing of publication-quality reports that
include chromatograms, method steps, and data tables
containing pertinent purification information, including yield,
concentration, and sample application and elution volumes
(Figure 1).
This study compares the performance and reproducibility of
the Profinia system to traditional manual gravity-flow column
purification for GST affinity purification and sequential
desalting. The B-PER GST fusion protein purification kit from
Pierce Biotechnology was chosen for comparison, as the
column volume (1 ml) is identical to the column volume used
by kits available for the Profinia protein purification system.
For the comparison, identical application and elution volumes
were used for the columns tested between the two systems,
further allowing a valid comparison. Desalting of the gravity-
flow column-purified protein requires manual application of
the protein eluted from the affinity column to a second column
for desalting, and this step was performed using Zeba desalt
spin columns from Pierce Biotechnology. These are available
with a column volume identical to the desalting column used
in the Profinia system (10 ml). The two purification methods ––
automated and manual –– were compared with respect to
yield, reproducibility, electrophoretic purity, total time required,
and hands-on time required.
chromatography
tech note 5513
Tom Berkelman and Michael Urban, Bio-Rad Laboratories Inc.,
Hercules, CA 94547 USA
Introduction
Fusion tags are widely used to assist in the purification of
recombinant protein expressed from a gene of interest. Among
the various fusion tags that have been developed, glutathione
S-transferase (GST) is one of the most commonly used (Smith
and Johnson 1988). Protein fused to GST can be purified from
crude bacterial lysates under nondenaturing conditions by
affinity chromatography on an immobilized glutathione column.
The lysate is applied to the column, the GST-tagged protein
binds to the immobilized glutathione, and unbound host
proteins are collected from the column in the flow-through
fraction. The column is then washed with several column
volumes of buffer to ensure complete removal of untagged
proteins. Purified protein is eluted and recovered in buffer
containing reduced glutathione, which displaces the GST-
tagged protein from the immobilized glutathione. This widely
performed procedure is often performed under gravity-flow
conditions, in which sample and buffers are applied onto an
open column and fractions are collected manually.
The Profinia protein purification system is an automated liquid
chromatography system designed to perform unattended
affinity purification and desalting of tagged proteins. Optimized
preprogrammed methods for the most common affinity
applications are available and can be used together with
the buffers and prepacked columns available in the Profinia
purification kits. All of the parameters for routine purifications
are preset, and the preplumbed system is easily maintained
through automated self-cleaning protocols. Integrated
collection of the flow-through, column washes, and elution