Bio-Rad Profinia Protein Purification Instrument User 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 gravity­flow 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 GST­tagged 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

© 2006 Bio-Rad Laboratories, Inc. Bulletin 5513

Results and Discussion

UV absorbance data from the Profinia purifications
demonstrate the performance of the automated GST +
Desalting method (Figure 2). The profiles of the UV traces at
the output of either column were virtually indistinguishable
from one another when compared using Profinia software.
The elution times for both the affinity and desalting peaks
varied by no more than 10 sec.

Fig. 2. Superimposed UV absorbance profiles from replicate GST + Desalting
purifications of the 51 kD protein on the Profinia system. The UV absorbance of

samples at 280 nm (AU) was collected over time for five consecutive purifications,
and the data are shown on the same pair of axes. ( ——), absorbance at the output
of the affinity column; ( ——), absorbance at the output of the desalting column.

Methods

A 51 kD GST-tagged protein expressed in Escherichia coli
was purified using both a Profinia system method and a
manual gravity-flow method for purification and desalting.
Five purification runs were performed, and samples of the
lysate, flow-through, wash, and purified protein fractions
from runs 1, 3, and 5 were then analyzed by SDS-PAGE.
The concentration of the purified 51 kD protein was determined
spectrophotometrically using the absorbance at 280 nm and
the known absorbance of a 1 mg/ml solution of the 51 kD
protein. Purity of the 51 kD protein was evaluated using the
Experion

™

automated electrophoresis system.

Protein Purification With the Profinia System

All purification reagents, columns, and a lyophilized E. coli
lysate containing the 51 kD GST-tagged protein were obtained
from the Profinia GST starter kit (catalog #620-0230). Each vial
of lysate was resuspended as directed in the kit instructions in
10 ml of Profinia lysis buffer. Purifications were performed with
the Profinia method template for “GST + Desalting” and with
the “low” sample flow rate. The 1 ml GST cartridge and 10 ml
desalting cartridge provided in the GST starter kit were used.
For each purification run, 5 ml of sample was loaded, which
required placement of 6 ml of lysate in the sample tube to
ensure complete loading of 5 ml of lysate. From each
purification run, 4 ml of purified and desalted protein was
collected. The five replicate runs were performed consecutively
using the same columns. Profinia software was used for real­time monitoring of the purification runs and collection of the
subsequent run data.

Protein Purification With the Pierce GST Fusion Protein Purification
Kit and Zeba Desalt Spin Columns

Each vial of E. coli lysate containing the 51 kD GST-tagged
protein from the Profinia GST starter kit was dissolved in 10 ml
of the B-PER bacterial protein extraction reagent provided in the
B-PER GST fusion protein purification kit (Pierce Biotechnology).
Purifications were carried out according to the supplied
instructions using an application volume of 5 ml and an elution
volume of 4 ml. Eluted protein from each GST purification was
desalted using 10 ml Zeba desalt spin columns (Pierce
Biotechnology) following the manufacturer’s instructions.
Five purification runs were performed using new columns for
each replicate.

SDS-PAGE and Experion Pro260 Analysis

SDS-PAGE analysis was performed using the Criterion™system
and 8–16% Tris-HCl precast gels. Gels were stained with
Bio-Safe

™

Coomassie stain. The sample, flow-through, and
wash fractions were diluted 10-fold into Laemmli sample buffer
and loaded in a volume of 10 µl. Purified protein was diluted in
Laemmli sample buffer to 0.1 µg/µl and loaded in a volume of
10 µl (1 µg). Experion analysis of the purified protein fractions
was performed using the Experion Pro260 analysis kit
following the protocol described in the instruction manual.

20 25 30 35 40 45 50

Run time, min

AU

2.0

1.8

1.6

1.4

1.2

1.0

0.8

0.6

0.4

0.2

0.0

Fig. 1. Example of a Profinia standard report. Profinia software prepares a report
containing chromatograms, continuous records of various method parameters,
protein yield, sample application and elution volumes, and purification conditions.

51 kD

affinity peak

51 kD

desalted peak