Purification of GST fusion proteins,
on-column cleavage and sample clean-up
Conclusions
Affinity tags such as GST can be used for many purposes, including one step
affinity purification. Two of the constructs described here held a protease
site next to the GST-tag to allow removal of the affinity tag from the target
protein. The use of PreScission protease gave pure GST-free protein in a
straightforward on-column reaction. After cleavage with thrombin, the
protease was easily removed with HiTrap Benzamidine FF (high sub)
connected directly to the GSTrap FF column.
Jill A. Sigrell, Amersham Biosciences AB, SE-751 84 Uppsala, Sweden.
Introduction
The use of tagged proteins is widely spread due to their many benefits, such as high
purity with only one step affinity purification, on-column refolding possibilities,
and easy detection. Glutathione Sepharose™ allows fast and easy protein purification
of recombinant proteins fused to the affinity tag Glutathione S-transferase (GST).
This affinity tag is widely used, and has the possibility to increase the expression
yield and the solubility and folding of the expressed protein. In this work, we show
the purification of six different human proteins all produced as fusions to GST
using different pGEX-vectors, and expressed in E. coli.
Acknowledgement
We thank Dr. Kristina Bäckbro (Uppsala University, Uppsala, Sweden) for GST-hippocalcin, Pharmacia Corporation
(Stockholm, Sweden) for SH2-GST and Martin Hammarström (Royal Institute of Technology, Stockholm, Sweden)
for the GST-fused ORFs.
Asia Pacific Tel: +852 2811 8693 Fax: +852 2811 5251 Australasia Tel: +61 2 9894 5152 Fax: +61 2 9899 7511 Austria Tel: 01 57 606 1620 Fax: 01 57 606 1627 Belgium Tel: 0800 73 888 Fax: 03 272 1637
Canada Tel: 1 800 463 5800 Fax: 1 800 567 1008 Central, East, South East Europe Tel: +43 1 982 3826 Fax: +43 1 985 8327 Denmark Tel: 45 16 2400 Fax: 45 16 2424 Finland Tel: 09 512 3940 Fax: 09 512 1710
France Tel: 0169 35 67 00 Fax: 0169 41 96 77 Germany Tel: 0761 4903 401 Fax: 0761 4903 405 Italy Tel: 02 27322 1 Fax: 02 27302 212 Japan Tel: 81 3 5331 9336 Fax: 81 3 5331 9370 Latin America Tel: +55 11 3667 5700
Fax: +55 11 3667 87 99 Middle East and Africa Tel: +30 (1) 96 00 687 Fax: +30 (1) 96 00 693 Netherlands Tel: 0165 580 410 Fax: 0165 580 401 Norway Tel: 23 18 5800 Fax: 23 18 6800 Portugal Tel: 01 417 7035
Fax: 01 417 3184 Russian Federation Tel: +7 (095) 232 0250,956 1137 Fax: +7 (095) 230 6377 South East Asia Tel: 60 3 724 2080 Fax: 60 3 724 2090 Spain Tel: 93 594 49 50 Fax: 93 594 49 55
Sweden Tel: +46 018 612 19 00 Fax: +46 018 612 19 10 Switzerland Tel: 01 802 81 50 Fax: 01 802 81 51 UK Tel: 0800 616 928 Fax: 0800 616 927 USA Tel: +1 800 526 3593 Fax: +1 800 329 3593
RAK design AB.
Sepharose, PreScission, ExcelGel, HiTrap, GSTrap and ÄKTA are trademarks of Amersham Pharmacia Biotech Limited or its subsidiaries. Amersham is a trademark of Nycomed Amersham plc. Pharmacia and Drop Design are
trademarks of Pharmacia Corporation. Coomassie is a trademark of ICI plc. Amersham Pharmacia Biotech AB Björkgatan 30, SE-751 84 Uppsala, Sweden. Amersham Pharmacia Biotech UK Limited Amersham Place, Little
Chalfont, Buckinghamshire HP7 9NA, England. Amersham Pharmacia Biotech Inc 800 Centennial Avenue, PO Box 1327, Piscataway, NJ 08855 USA. Amersham Pharmacia Biotech Europe GmbH Munzinger Strasse 9,
D-79111 Freiburg, Germany. Amersham Pharmacia Biotech K.K. Sanken Building, 3-25-1, Hyakunincho, Shinjuku-ku, Tokyo 169-0073, Japan. All goods and services are sold subject to the terms and conditions of sale of
the company within the Amersham Pharmacia Biotech group that supplies them. A copy of these terms and conditions is available on request. © Amersham Pharmacia Biotech AB 2001 – All rights reserved.
18-1150-20
Edition AA
Elution of
GSTrap FF
On-column cleavage using PreScission protease
Columns:
Binding and wash buffer:
GST elution buffer:
Flow rate:
Instrument:
Protease treatment:
2 ml clarified E. coli homogenate containing expressed
GST-hippocalcin, Mr 43 000
2 x GSTrap FF, 1 ml
50 mM Tris-HCl pH 8.0, 0.15 M NaCl, 1 mM CaCl2, 1 mM DTT,
10% glycerol
50 mM Tris-HCl pH 8.0, 20 mM reduced glutathione
0.5 ml/min
ÄKTA™prime
80 U/ml PreScission protease overnight at +4 °C and at room
temperature for 2 hours
0.80
GST-tag and
PreScission
protease
PreScission Continued
protease column wash
Column
wash
0.20
Lane 1:
Lane 2:
Lane 3:
Lane 4:
Lane 5:
Lane 6:
20
30
40
ml
2 x GSTrap FF
High salt
buffer wash
Gel: ExcelGel SDS Gradient 8-18%, Coomassie staining
Elution of
GSTrap FF
A 280 nm
Thrombin activity
A 405 nm
GST-tag
0.30
97 000
66 000
45 000
0.20
30 000
20 100
14 400
0.60
Thrombin
Column
wash
0.40
0.20
0.10
fr.14
fr.6
fr.7
fr.8
fr.2
fr.21
fr.22
SH2
0
0
10
0
A)
15
A)
20
25
50
B)
ml
A)
A) GSTrap FF
B) HiTrap Benzamidine FF (high sub)
B)
Fig 3. Chromatogram and activity curve demonstrating all steps in the purification of
the SH2 domain.
Column:
Binding buffer:
GST elution buffer:
Flow rate:
Instrument:
3
4
5
6
7 8
9
0.80
A)
Clarified E. coli homogenates containing expressed
GST-tagged ORFs
GSTrap FF, 1 ml
20 mM Na phosphate, pH 7.5, 0.15 M NaCl
50 mM Tris-HCl pH 8.0, 20 mM reduced glutathione
0.5 ml/min
ÄKTAprime
1 2
Mr
Sample:
Sample:
Clarified E. coli homogenate containing expressed GST-hippocalcin
Flow through (fraction 2)
GST-hippocalcin
Pure hippocalcin after on-column cleavage (fraction 5)
Same as lane 4, but fraction 6
Eluted fraction from GSTrap FF containing PreScission protease, and GST-tag
released by cleavage (fraction 12)
Fig 2. SDS PAGE analysis.
Elution of HiTrap
Benzamidine FF (high sub)
Thrombin
Material and Methods
GST-hippocalcin
PreScission protease
GST
hippocalcin
Hippocalcin
10
On-column cleavage using thrombin
As part of a structural genomics project, where the aim is
to determine 3D structures of proteins at high speed, open
reading frames (ORFs) were cloned as GST-fusions. The
proteins were first produced and purified in a small scale.
Using a pre-programmed template on ÄKTAprime, the
purification of these four proteins from cell extracts was
quickly performed, making it possible to evaluate easily each
protein's expression, purity and stability. The results seen
in Figure 5 show good protein expression. However, a clear
degradation pattern was observed for all the purified proteins.
6
20 100
14 400
GSTrap FF
Initial purification screen within a structural genomics program
5
30 000
0.40
0
2 ml clarified E. coli homogenate containing expressed SH2-GST,
Mr 37 000
Columns:
GSTrap FF, 1 ml and HiTrap Benzamidine FF (high sub), 1 ml
Binding buffer:
20 mM Na phosphate, pH 7.5, 0.15 M NaCl
High salt wash buffer:
20 mM Na phosphate, pH 7.5, 1.0 M NaCl
Benzamidine elution buffer: 20 mM p-aminobenzamidine in binding buffer
GST elution buffer:
20 mM reduced glutathione, 50 mM Tris, pH 8.0
Flow rate:
0.5 ml/min
Instrument:
ÄKTAprime
20 U/ml thrombin (Amersham Biosciences) for 2 hours
Protease treatment:
at room temperature
Thrombin activity:
S-2238 (Haemochrom Diagnostica AB) was used as a substrate
and its absorbance at 405 nm was measured
4
45 000
0.60
0
Material and Methods
3
97 000
66 000
Fig 1. Chromatogram showing the purification of human hippocalcin.
When expressing the SH2 domain from a phosphatase, a
fusion protein containing a thrombin site between the SH2
domain and the GST-tag was created. Thrombin (a serine
protease that can bind the synthetic inhibitor p-aminobenzamidine) could be completely removed after on-column
cleavage by placing a HiTrap™ Benzamidine FF (high sub)
column in series after the GSTrap FF column. The removal
of thrombin was verified with an activity assay using the
substrate S-2238. From one gram of wet cells, 5 mg of
pure tag-free SH2 domain was obtained.
2
Mr
fr.12
Sample:
1
A 280 nm
fr.5
fr.6
Material and Methods
fr.2
The gene for human hippocalcin, a member of the neuronespecific calcium-binding protein family, was incorporated
in a pGEX-vector holding a PreScission™ protease site
next to the GST-tag. After capturing the expressed fusion
protein on a GSTrap™ FF column, PreScission protease
(which is GST-tagged itself) was added. The on-column
cleavage reaction took place at +4 °C overnight and at
room temperature for an additional 2 hours. To recover
hippocalcin, another GSTrap FF column was placed in
series after the first, to make sure no PreScission protease,
uncleaved GST-fusion or free GST-tag came through with
the sample during the additional wash with binding buffer.
For every gram of wet E. coli cells, 10 mg of pure, nontagged hippocalcin was obtained.
Gel: ExcelGel™ SDS Gradient, 8-18%, Coomassie™ staining
SH2
Lane 1.
Lane 2.
Lane 3.
Lane 4.
Lane 5.
Lane 6.
Lane 7.
Low Molecular Weight marker
Clarified E. coli homogenate containing expressed SH2-GST
Flow through fraction (fraction 2)
SH2 domain, washed off with binding buffer through both columns (fraction 6)
Same as lane 4, but fraction 7
Same as lane 4, but fraction 8
Eluted fraction from HiTrap Benzamidine FF (high sub) with the highest
thrombin activity (fraction 14)
Lane 8. Eluted fraction from GSTrap FF containing cleaved off GST-tag and
un-cleaved fusion protein, SH2-GST (fraction 21)
Lane 9. Same as lane 8, but fraction 22
Fig 4. SDS PAGE anaysis.
Gel: ExcelGel SDS Gradient 8-18%, Coomassie staining
Clone A, Mr 37 000
S
FT
E
Clone B, Mr 36 000
Mr
Mr
S
FT
E
Clone C, Mr 39 000
Mr
Mr
S
FT
E
Clone D, Mr 36 000
Mr
Mr
S
FT
E
Mr
Mr
97 000
66 000
97 000
66 000
97 000
66 000
97 000
66 000
45 000
45 000
45 000
45 000
30 000
30 000
30 000
20 100
14 400
20 100
14 400
20 100
14 400
30 000
20 100
14 400
Fig 5. SDS PAGE analysis of 4 different human open reading frames (A-D) expressed as GST-fusions. S) sample loaded on a GSTrap FF column, FT) flow through
during sample load and wash, E) eluted fusion protein and Mr) low molecular weight marker proteins.
Elution of
GSTrap FF
On-column cleavage using PreScission protease
Columns:
Binding and wash buffer:
GST elution buffer:
Flow rate:
Instrument:
Protease treatment:
2 ml clarified E. coli homogenate containing expressed
GST-hippocalcin, Mr 43 000
2 x GSTrap FF, 1 ml
50 mM Tris-HCl pH 8.0, 0.15 M NaCl, 1 mM CaCl2, 1 mM DTT,
10% glycerol
50 mM Tris-HCl pH 8.0, 20 mM reduced glutathione
0.5 ml/min
ÄKTA™prime
80 U/ml PreScission protease overnight at +4 °C and at room
temperature for 2 hours
0.80
GST-tag and
PreScission
protease
PreScission Continued
protease column wash
Column
wash
0.20
Lane 1:
Lane 2:
Lane 3:
Lane 4:
Lane 5:
Lane 6:
20
30
40
ml
2 x GSTrap FF
High salt
buffer wash
Gel: ExcelGel SDS Gradient 8-18%, Coomassie staining
Elution of
GSTrap FF
A 280 nm
Thrombin activity
A 405 nm
GST-tag
0.30
97 000
66 000
45 000
0.20
30 000
20 100
14 400
0.60
Thrombin
Column
wash
0.40
0.20
0.10
fr.14
fr.6
fr.7
fr.8
fr.2
fr.21
fr.22
SH2
0
0
10
0
A)
15
A)
20
25
50
B)
ml
A)
A) GSTrap FF
B) HiTrap Benzamidine FF (high sub)
B)
Fig 3. Chromatogram and activity curve demonstrating all steps in the purification of
the SH2 domain.
Column:
Binding buffer:
GST elution buffer:
Flow rate:
Instrument:
3
4
5
6
7 8
9
0.80
A)
Clarified E. coli homogenates containing expressed
GST-tagged ORFs
GSTrap FF, 1 ml
20 mM Na phosphate, pH 7.5, 0.15 M NaCl
50 mM Tris-HCl pH 8.0, 20 mM reduced glutathione
0.5 ml/min
ÄKTAprime
1 2
Mr
Sample:
Sample:
Clarified E. coli homogenate containing expressed GST-hippocalcin
Flow through (fraction 2)
GST-hippocalcin
Pure hippocalcin after on-column cleavage (fraction 5)
Same as lane 4, but fraction 6
Eluted fraction from GSTrap FF containing PreScission protease, and GST-tag
released by cleavage (fraction 12)
Fig 2. SDS PAGE analysis.
Elution of HiTrap
Benzamidine FF (high sub)
Thrombin
Material and Methods
GST-hippocalcin
PreScission protease
GST
hippocalcin
Hippocalcin
10
On-column cleavage using thrombin
As part of a structural genomics project, where the aim is
to determine 3D structures of proteins at high speed, open
reading frames (ORFs) were cloned as GST-fusions. The
proteins were first produced and purified in a small scale.
Using a pre-programmed template on ÄKTAprime, the
purification of these four proteins from cell extracts was
quickly performed, making it possible to evaluate easily each
protein's expression, purity and stability. The results seen
in Figure 5 show good protein expression. However, a clear
degradation pattern was observed for all the purified proteins.
6
20 100
14 400
GSTrap FF
Initial purification screen within a structural genomics program
5
30 000
0.40
0
2 ml clarified E. coli homogenate containing expressed SH2-GST,
Mr 37 000
Columns:
GSTrap FF, 1 ml and HiTrap Benzamidine FF (high sub), 1 ml
Binding buffer:
20 mM Na phosphate, pH 7.5, 0.15 M NaCl
High salt wash buffer:
20 mM Na phosphate, pH 7.5, 1.0 M NaCl
Benzamidine elution buffer: 20 mM p-aminobenzamidine in binding buffer
GST elution buffer:
20 mM reduced glutathione, 50 mM Tris, pH 8.0
Flow rate:
0.5 ml/min
Instrument:
ÄKTAprime
20 U/ml thrombin (Amersham Pharmacia Biotech) for 2 hours
Protease treatment:
at room temperature
Thrombin activity:
S-2238 (Haemochrom Diagnostica AB) was used as a substrate
and its absorbance at 405 nm was measured
4
45 000
0.60
0
Material and Methods
3
97 000
66 000
Fig 1. Chromatogram showing the purification of human hippocalcin.
When expressing the SH2 domain from a phosphatase, a
fusion protein containing a thrombin site between the SH2
domain and the GST-tag was created. Thrombin (a serine
protease that can bind the synthetic inhibitor p-aminobenzamidine) could be completely removed after on-column
cleavage by placing a HiTrap™ Benzamidine FF (high sub)
column in series after the GSTrap FF column. The removal
of thrombin was verified with an activity assay using the
substrate S-2238. From one gram of wet cells, 5 mg of
pure tag-free SH2 domain was obtained.
2
Mr
fr.12
Sample:
1
A 280 nm
fr.5
fr.6
Material and Methods
fr.2
The gene for human hippocalcin, a member of the neuronespecific calcium-binding protein family, was incorporated
in a pGEX-vector holding a PreScission™ protease site
next to the GST-tag. After capturing the expressed fusion
protein on a GSTrap™ FF column, PreScission protease
(which is GST-tagged itself) was added. The on-column
cleavage reaction took place at +4 °C overnight and at
room temperature for an additional 2 hours. To recover
hippocalcin, another GSTrap FF column was placed in
series after the first, to make sure no PreScission protease,
uncleaved GST-fusion or free GST-tag came through with
the sample during the additional wash with binding buffer.
For every gram of wet E. coli cells, 10 mg of pure, nontagged hippocalcin was obtained.
Gel: ExcelGel™ SDS Gradient, 8-18%, Coomassie™ staining
SH2
Lane 1.
Lane 2.
Lane 3.
Lane 4.
Lane 5.
Lane 6.
Lane 7.
Low Molecular Weight marker
Clarified E. coli homogenate containing expressed SH2-GST
Flow through fraction (fraction 2)
SH2 domain, washed off with binding buffer through both columns (fraction 6)
Same as lane 4, but fraction 7
Same as lane 4, but fraction 8
Eluted fraction from HiTrap Benzamidine FF (high sub) with the highest
thrombin activity (fraction 14)
Lane 8. Eluted fraction from GSTrap FF containing cleaved off GST-tag and
un-cleaved fusion protein, SH2-GST (fraction 21)
Lane 9. Same as lane 8, but fraction 22
Fig 4. SDS PAGE anaysis.
Gel: ExcelGel SDS Gradient 8-18%, Coomassie staining
Clone A, Mr 37 000
S
FT
E
Clone B, Mr 36 000
Mr
Mr
S
FT
E
Clone C, Mr 39 000
Mr
Mr
S
FT
E
Clone D, Mr 36 000
Mr
Mr
S
FT
E
Mr
Mr
97 000
66 000
97 000
66 000
97 000
66 000
97 000
66 000
45 000
45 000
45 000
45 000
30 000
30 000
30 000
20 100
14 400
20 100
14 400
20 100
14 400
30 000
20 100
14 400
Fig 5. SDS PAGE analysis of 4 different human open reading frames (A-D) expressed as GST-fusions. S) sample loaded on a GSTrap FF column, FT) flow through
during sample load and wash, E) eluted fusion protein and Mr) low molecular weight marker proteins.
Purification of GST fusion proteins,
on-column cleavage and sample clean-up
Conclusions
Affinity tags such as GST can be used for many purposes, including one step
affinity purification. Two of the constructs described here held a protease
site next to the GST-tag to allow removal of the affinity tag from the target
protein. The use of PreScission protease gave pure GST-free protein in a
straightforward on-column reaction. After cleavage with thrombin, the
protease was easily removed with HiTrap Benzamidine FF (high sub)
connected directly to the GSTrap FF column.
Jill A. Sigrell, Amersham Pharmacia Biotech AB, SE-751 84 Uppsala, Sweden.
[email protected]
Introduction
The use of tagged proteins is widely spread due to their many benefits, such as high
purity with only one step affinity purification, on-column refolding possibilities,
and easy detection. Glutathione Sepharose™ allows fast and easy protein purification
of recombinant proteins fused to the affinity tag Glutathione S-transferase (GST).
This affinity tag is widely used, and has the possibility to increase the expression
yield and the solubility and folding of the expressed protein. In this work, we show
the purification of six different human proteins all produced as fusions to GST
using different pGEX-vectors, and expressed in E. coli.
Acknowledgement
We thank Dr. Kristina Bäckbro (Uppsala University, Uppsala, Sweden) for GST-hippocalcin, Amersham Biosciences
(Stockholm, Sweden) for SH2-GST and Martin Hammarström (Royal Institute of Technology, Stockholm, Sweden)
for the GST-fused ORFs.
Asia Pacific Tel: +852 2811 8693 Fax: +852 2811 5251 Australasia Tel: +61 2 9894 5152 Fax: +61 2 9899 7511 Austria Tel: 01 57 606 1620 Fax: 01 57 606 1627 Belgium Tel: 0800 73 888 Fax: 03 272 1637
Canada Tel: 1 800 463 5800 Fax: 1 800 567 1008 Central, East, South East Europe Tel: +43 1 982 3826 Fax: +43 1 985 8327 Denmark Tel: 45 16 2400 Fax: 45 16 2424 Finland Tel: 09 512 3940 Fax: 09 512 1710
France Tel: 0169 35 67 00 Fax: 0169 41 96 77 Germany Tel: 0761 4903 401 Fax: 0761 4903 405 Italy Tel: 02 27322 1 Fax: 02 27302 212 Japan Tel: 81 3 5331 9336 Fax: 81 3 5331 9370 Latin America Tel: +55 11 3667 5700
Fax: +55 11 3667 87 99 Middle East and Africa Tel: +30 (1) 96 00 687 Fax: +30 (1) 96 00 693 Netherlands Tel: 0165 580 410 Fax: 0165 580 401 Norway Tel: 23 18 5800 Fax: 23 18 6800 Portugal Tel: 01 417 7035
Fax: 01 417 3184 Russian Federation Tel: +7 (095) 232 0250,956 1137 Fax: +7 (095) 230 6377 South East Asia Tel: 60 3 724 2080 Fax: 60 3 724 2090 Spain Tel: 93 594 49 50 Fax: 93 594 49 55
Sweden Tel: +46 018 612 19 00 Fax: +46 018 612 19 10 Switzerland Tel: 01 802 81 50 Fax: 01 802 81 51 UK Tel: 0800 616 928 Fax: 0800 616 927 USA Tel: +1 800 526 3593 Fax: +1 800 329 3593
RAK design AB.
Sepharose, PreScission, ExcelGel, HiTrap, GSTrap and ÄKTA are trademarks of Amersham Biosciences Limited or its subsidiaries. Amersham is a trademark of Nycomed Amersham plc.
Coomassie is a trademark of ICI plc. Amersham Biosciences AB Björkgatan 30, SE-751 84 Uppsala, Sweden. Amersham Biosciences UK Limited Amersham Place, Little
Chalfont, Buckinghamshire HP7 9NA, England. Amersham Biosciences Inc 800 Centennial Avenue, PO Box 1327, Piscataway, NJ 08855 USA. Amersham Biosciences Europe GmbH Munzinger Strasse 9,
D-79111 Freiburg, Germany. Amersham Biosciences K.K. Sanken Building, 3-25-1, Hyakunincho, Shinjuku-ku, Tokyo 169-0073, Japan. All goods and services are sold subject to the terms and conditions of sale of
the company within the Amersham Biosciences group that supplies them. A copy of these terms and conditions is available on request. © Amersham Biosciences AB 2001 – All rights reserved.
18-1150-20
Edition AA