GE Healthcare
Application Note 28-9339-91 AA
Chromatography systems
Unattended affinity purification and buffer
exchange on ÄKTAprime plus
Abstract
A
We describe a new protocol for affinity purification followed
by immediate buffer exchange on ÄKTAprime™ plus
chromatography system. The protocol proved to be very
effective when tested on three tagged proteins and two
antibodies. Reverse flow elution and direct transfer of the
proteins from an affinity column onto desalting columns
for buffer exchange reduced peak dilution, saved time and
eliminated manual involvement.
Chromatograms and SDS-PAGE results revealed sharp peaks
and high final purities for all five protein samples. Up to 40 mg
of protein can be purified in about 80 minutes. This new protocol
is applicable to any affinity purification on ÄKTAprime plus
where the affinity column volume is one ml and the total
volume of the desalting columns is 10 ml.
Introduction
1
Equilibration of desalting columns
for buffer exchange
Pump
2
7
UV
6
3
5
4
Equilibration, sample load and
wash of affinity column
Waste
Position 1 - Load
Switching of the injection valve to
connect desalting columns at the
top end of the affinity column
Equilibration, sample application and
wash of affinity column
1
Pump
Elution of affinity column in reverse
flow mode and buffer exchange
over the desalting columns
ÄKTAprime plus is an automated liquid chromatography
system for laboratory-scale purifications. The system features
preprogrammed methods for all common chromatographic
techniques.
Affinity chromatography continues to increase in popularity
as a purification technique when purifying tagged proteins
and antibodies. However, the elution buffer conditions (pH, salt
composition and additives) in which target proteins elute can
be detrimental to the proteins. To maintain their structural
and functional integrity, therefore, proteins should be
transferred to gentler conditioning buffers without delay.
B
2
7
UV
6
3
5
4
Waste
Elution of purified protein in
conditioning buffer
Position 2 - Inject
Equilibration of buffer exchange column
and elution from affinity column directly
onto the desalting column
C
In addition, buffer exchange of proteins in general is often
necessary prior to the next chromatography step. Here again,
the need for immediate transfer to a more suitable buffer or
storage is clear.
Fig 1. A) Workflow for the new method file for unattended affinity purification/
buffer exchange. B) Configuration of the ÄKTAprime plus injection valve for
the application. C) System setup during the application.
imagination at work
Experimental set-up
To meet this need for ‘instantaneous’ buffer exchange, we
have developed a new protocol for unattended affinity
purification followed by immediate buffer exchange. This
Application Note illustrates the use of this new protocol for
tagged protein and antibody purifications.
Figure 1 shows the workflow and the injection valve configuration
for the unattended affinity purification/buffer exchange on
ÄKTAprime plus. The mixer was disconnected from the system
during this procedure. Full details of how to configure the
system to run this method are available on Cue Card.
Goals
Samples and columns
Our main goal was to develop a new method file for
ÄKTAprime plus that allows transfer of peaks eluted from an
affinity column directly onto a column for buffer exchange,
to save time and eliminate manual intervention. In addition,
the new protocol should be generally applicable to any type
of affinity purification.
Materials and methods
Unless otherwise stated, all equipment and chromatographic
media were from GE Healthcare (Uppsala, Sweden) and all
chemicals used were of analytical grade.
Tagging proteins simplifies purification, and some of the most
common affinity tags include polyhistidine (His), glutathione
S-transferase (GST), maltose-binding protein (MBP), and
streptavidin-binding peptide (Strep II). Although the basic
purification protocol is the same for all, binding and elution
conditions differ. Polyhistidine tagged proteins are eluted in
high concentrations of imidazole, whereas GST-tagged proteins
elute in glutathione, for example.
Three tagged proteins and two antibodies were purified and
buffer exchanged using the new protocol. Table 1 lists the
columns and sample proteins used. Two HiTrap Desalting
columns were connected in series to increase the bed volume
to 10 ml.
Table 1. Columns and samples used for purifications
Tagged protein
purification
Antibody
purification
Buffer exchange
Column
Medium
Sample
Tags used
HisTrap FF crude 1 ml
Ni Sepharose 6 Fast Flow
DC44-(His)8
Polyhistidine (His)
MBPTrap™ HP 1 ml
Dextrin Sepharose
High Performance
MBP2*-paramyosindeltaSal
Maltose-binding protein
(MBP)
GSTrap™ FF 1 ml
Glutathione Sepharose
Fast Flow
GST-Hippocalcin
Glutathione-S-transferase
(GST)
HiTrap™ Protein A HP
Protein A Sepharose
High Performance
IgG1 from mouse
-
HiTrap Protein G HP
Protein A Sepharose
High Performance
IgG2a from mouse
-
HiTrap Desalting 5 ml
Sephadex™ G25
Eluted protein from
affinity column
-
™
™
01/2008 28-9339-91 AA
2
Tagged protein purification and buffer exchange
Three tagged proteins were purified by affinity chromatography
and buffer exchanged immediately using the new method
file for ÄKTAprime plus. For purifying histidine-tagged
protein, HisTrap FF crude, a column specially designed for
applying unclarified sample, was used. The results from the
purifications, including chromatographic conditions, are
shown in Figure 2 to 4.
Purification of MBP-tagged protein
Affinity purification
Column:
MBPTrap HP 1 ml
Sample:
0.75 ml MBP2*-paramyosin-deltaSal (Mr ~70 200) in clarified
E. coli lysate. Volume adjusted to 20 ml in binding buffer
prior to loading.
Flow rate:
1 ml/min
Binding buffer: 20 mM Tris-HCl, 200 mM NaCl, 1 mM EDTA, 1 mM DTT, pH 7.4
Elution buffer:
10 mM maltose in binding buffer
Buffer exchange
Column:
2 × HiTrap Desalting 5 ml
Purification of histidine-tagged protein
Affinity purification
Sample:
Eluted pool from MBPTrap HP 1 ml
Flow rate:
3 ml/min
Column: HisTrap FF crude 1 ml
Buffer:
20 mM sodium phosphate, 150 mM NaCl, pH 7.0
Sample: 20 ml DC44-(His)8 (Mr ~16 000) in unclarified E. coli lysate
Flow rate: 1 ml/min
A
Binding buffer: 20 mM sodium phosphate, 0.5 M NaCl, 35 mM imidazole, pH 7.4
800
2 × HiTrap Desalting 5 ml
Sample: Eluted pool from HisTrap FF crude 1 ml
Flow rate: 3 ml/min
Buffer: 20 mM sodium phosphate, 150 mM NaCl, pH 7.0
600
10
400
Valve-switch
Buffer exchange
15
Valve-switch
UV 280 nm mAu
Elution buffer: 20 mM sodium phosphate, 0.5 M NaCl, 0.5 M imidazole, pH 7.4
Column:
20
1000
200
5
0
0
3000
50
10
20
30
2000
30
1500
Valve-switch
20
1000
500
0
0
10
20
30
40
50
60
70
B
50
60
70
min
Mr × 103
97.0 66.0 -
10
45.0 -
0
30.0 -
min
20.1 1. Low Molecular Weight
2. Sample
3. Flowthrough
4. Eluted peak
14.4 -
B
40
40
Valve-switch
UV 280 nm mAu
2500
Cond. mS/cm
A
0
Cond. mS/cm
Results
1
3
Mr × 10
2
3
4
Fig 3. Unattended affinity purification and buffer exchange of MBP2*paramyosin-deltaSal on ÄKTAprime plus. A) Chromatogram of the
purification. UV detection at 280 nm. B) SDS-PAGE analysis. Reduced
conditions on ExcelGel SDS Gradient 8-18. Coomassie stained.
97.0 66.0 45.0 -
30.0 20.1 -
1. Low Molecular Weight
2. Sample
3. Flowthrough
4. Eluted peak
14.4 1
2
3
4
Fig 2. Unattended affinity purification and buffer exchange of DC44-(His)8 on
ÄKTAprime plus. A) Chromatogram of the purification. UV detection at 280 nm.
B) SDS-PAGE analysis. Reduced conditions on ExcelGel™ SDS Gradient 8-18.
Coomassie™ stained.
01/2008 28-9339-91 AA
3
Antibody purification and buffer exchange
Purification of GST-tagged protein
Affinity purification
Column:
GSTrap FF 1 ml
Sample:
5 ml GST-Hippocalcin (Mr ~45 000) in clarified E. coli lysate.
Volume adjusted to 20 ml in binding buffer prior to loading.
Flow rate:
1 ml/min
Binding buffer: PBS
Elution buffer:
50 mM Tris-HCl, 10 mM reduced glutathione, pH 8.0
Two different antibodies were purified by affinity
chromatography and buffer exchanged immediately using
the new method file for ÄKTAprime plus. The results from
the purifications, including chromatographic conditions, are
shown in Figures 5 and 6
Buffer exchange
Column:
2 × HiTrap Desalting 5 ml
Sample:
Eluted pool from GSTrap™ FF 1 ml
Flow rate:
3 ml/min
Buffer:
20 mM sodium phosphate, 150 mM NaCl, pH 7.0
16
A
HiTrap Protein G HP 1 ml
Sample:
3 ml monoclonal mouse IgG1 in cell culture supernatant.
Volume adjusted to 20 ml in binding buffer prior to loading.
Flow rate:
1 ml/min
8
6
Valve-switch
1000
10
20
30
40
50
60
70
Binding buffer: 20 mM sodium phosphate, pH 7.0
Elution buffer:
0.1 M glycine HCl, pH 2.7
4
2
0
0
0
Cond. mS/cm
Column:
12
1500
Valve-switch
UV 280 nm mAu
14
10
500
B
Affinity purification
2500
2000
Protein A Sepharose HP and Protein G Sepharose HP are
commonly used affinity media for purifying antibodies, which
bind at neutral pH and elute at low pH. Elution at acidic pH,
however, may cause aggregation, conformational changes or
proteolytic cleavage of the target antibody. Immediate buffer
exchange will minimize such risks.
min
Buffer exchange
Column:
2 × HiTrap Desalting 5 ml
Sample:
Eluted pool from HiTrap Protein G HP 1 ml
Flow rate:
3 ml/min
Buffer:
20 mM sodium phosphate, 150 mM NaCl, pH 7.0
Mr × 103
A
8
700
97.0 -
7
600
30.0 -
500
6
pH
400
5
300
200
20.1 -
1. Low Molecular Weight
2. Sample
3. Flowthrough
4. Eluted peak
14.4 1
2
3
100
4
3
0
4
Fig 4. Unattended affinity purification and buffer exchange of GST-Hippocalcin
on ÄKTAprime plus. A) Chromatogram of the purification. UV detection at
280 nm. B) SDS-PAGE analysis. Reduced conditions on ExcelGel SDS Gradient
8-18. Coomassie stained.
Valve-switch
45.0 -
Valve-switch
UV 280 nm mAu
66.0 -
0
B
10
20
30
40
50
60
70
min
Mr × 103
97.0 66.0 45.0 30.0 20.1 1. Low Molecular Weight
2. Sample
3. Flowthrough
4. Eluted peak
14.4 -
1
2
3
4
Fig 5. Unattended affinity purification and buffer exchange of mouse IgG1 on
ÄKTAprime plus. A) Chromatogram of the purification. UV detection at 280 nm.
B) SDS-PAGE analysis. Reduced conditions on ExcelGel SDS Gradient 8-18.
Coomassie stained.
01/2008 28-9339-91 AA
4
Discussion
Affinity purification
Column:
HiTrap Protein A HP 1 ml
Sample:
20 ml monoclonal mouse IgG2a in cell culture supernatant
Flow rate:
1 ml/min
Binding buffer: 100 mM sodium phosphate, 100 mM sodium citrate, pH 7.0
Elution buffer:
100 mM sodium phosphate, 100 mM sodium citrate, pH 2.7
Buffer exchange
Column:
2 × HiTrap Desalting 5 ml
Sample:
Eluted pool from HiTrap Protein A HP 1 ml
Flow rate:
3 ml/min
Buffer:
20 mM sodium phosphate, 150 mM NaCl, pH 7.0
A
8
7
6
Valve-switch
5
500
4
3
0
0
B
pH
7
1000
Valve-switch
UV 280 nm mAu
1500
10
20
30
40
50
60
70
min
3
Mr × 10
97.0 66.0 45.0 30.0 20.1 1. Low Molecular Weight
2. Sample
3. Flowthrough
4. Eluted peak
14.4 -
1
2
3
4
Fig 6. Unattended affinity purification and buffer exchange of mouse IgG2a on
ÄKTAprime plus. A) Chromatogram of the purification. UV detection at 280 nm.
B) SDS-PAGE analysis. Reduced conditions on ExcelGel SDS Gradient 8-18.
Coomassie stained.
This unattended affinity purification/buffer exchange protocol
saved time, improved reproducibility and reduced manual
involvement.
The chromatogram and the SDS-PAGE analysis of the eluted
peaks revealed narrow elution peaks plus consistently high
purity. Eluting the affinity column using reverse flow and
transferring the eluted proteins directly to the buffer exchange
columns reduces dilution and gives a very sharp peak.
The total run-time from the equilibration to elution for a
protocol was about 80 minutes. The method offers the possibility
of changing the sample volume as well as volume of wash
buffer. In all the purifications presented, after loading 20 ml of
sample, the column was washed with 35 ml of binding buffer.
From the chromatography curves, it is observed that 10 ml of
wash with binding buffer often is sufficient to reach the base
line level. Hence one can significantly reduce the overall runtime to 50-60 minutes by decreasing the wash volume and
even more if sample volume is less.
Conclusions
The new ÄKTAprime plus protocol for affinity purification
followed by immediate buffer exchange performed well. For
the tagged recombinant protein and antibody purifications
described above, the goal of unattended ‘instantaneous’
buffer exchange was met. The proteins of interest eluted in
narrow peak volumes, and purities, as judged by SDS-PAGE
analysis, were high. Furthermore, unattended operation made
the method fast and convenient.
Finally, the protocol is applicable to any affinity purification
as long as the volume of the affinity column is one ml and
the total volume of the desalting columns is 10 ml. As a wide
range of affinity columns with this bed volume is available,
the new protocol should find widespread use.
Acknowledgements
Protein DC44-His8 used in this study was obtained through
cooperation with Biovitrum AB, SE-112 76 Stockholm, Sweden.
Ordering information
The kit including method file, tubing, connectors and Cue Card
for unattended purification using ÄKTAprime plus can be
ordered from GE Healthcare on 28-9348-28. The kit also
contains methods that allow unattended one step affinity
purification for two different samples using step or gradient
elution.
01/2008 28-9339-91 AA
5
GE, imagination at work and GE monogram are trademarks of General Electric Company.
Drop design, ÄKTAprime, ExcelGel, GSTrap, HisTrap, HiTrap, MBPTrap, Sephadex and
Sepharose are trademarks of GE Healthcare companies.
For contact information for your local office,
please visit, www.gelifesciences.com/contact
Purification and preparation of fusion proteins and affinity peptides comprising at
least two adjacent histidine residues may require a license under US patent numbers
5,284,933 and 5,310,663 and equivalent patents and patent applications in other
countries (assignee: Hoffman La Roche, Inc).
www.gelifesciences.com/akta
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Sweden
All goods and services are sold subject to the terms and conditions of sale of the
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conditions is available on request. Contact your local GE Healthcare representative
for the most current information
© 2008 General Electric Company – All rights reserved.
First published Jan. 2008.
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NJ 08855-1327, USA
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imagination at work
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Tokyo, 169-0073 Japan
28-9339-91 AA 01/2008
Elanders i Uppsala 2008
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