Download Fast and simple purification of GST fusion proteins using prepacked

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Fast and simple purification of GST fusion proteins
using prepacked GSTrap affinity columns
L. Haneskog, H. J. Johansson, and A. Heijbel
Amersham Biosciences, Uppsala, Sweden
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ready-to-use 1 ml and 5 ml HiTrap columns containing Glutathione Sepharose 4 Fast Flow (Fig 1). The
columns are designed for one-step purification of GST
fusion proteins, other glutathione S-transferases, and
glutathione binding proteins. Sample application,
washing, and elution can be performed using a
syringe, pump, or liquid chromatography system such
as ÄKTA™design, GradiFrac™, or FPLC™.
GSTrap™ affinity columns are prepacked, ready-to-use
1 ml and 5 ml HiTrap™ columns containing Glutathione
Sepharose™ 4 Fast Flow. They are designed for one-step
purification of glutathione S-transferase (GST) fusion
proteins, other glutathione S-transferases, and
glutathione binding proteins. Sample application,
washing, and elution can be performed using a syringe,
pump, or liquid chromatography system. Here, we
demonstrate the performance of the columns by
purifying a GST fusion protein from 8 ml and 40 ml
of a cell lysate using GSTrap 1 ml and 5 ml columns,
respectively. On-column cleavage with thrombin is
also demonstrated.
Introduction
The Glutathione S-transferase (GST) Gene Fusion
System is a versatile expression system that has been
cited in thousands of published studies involving
protein-protein interactions, nucleic acid-protein
interactions, antibody production, and many other
applications. The system is based on inducible, highlevel, bacterial expression of genes or gene fragments
as fusions with Schistosoma japonicum GST (1). GST
fusion proteins are constructed by inserting a gene or
gene fragment into the multiple cloning site of one of
the 13 pGEX vectors. Expression in E. coli yields
fusion proteins with the 26 kDa GST moiety at the
amino terminus and the protein of interest at the
carboxyl terminus.
Fig 1. GSTrap and Glutathione Sepharose 4 Fast Flow are designed for
the isolation and purification of GST fusion proteins, other glutathione
S-transferases and glutathione binding proteins.
Column design
HiTrap columns are convenient, reliable, and affordable multi-use columns designed for fast and easy
preparative separations. The columns can be used
separately or connected together in series. Columns
are made of medical-grade polypropylene, a biocompatible material that does not interact with biomolecules. Top and bottom frits are manufactured from
porous polyethylene. The frits allow high flow rates
but prevent columns from running dry. GSTrap
columns are delivered with a stopper on the inlet
and a twist-off end on the outlet. Both ends have
M6 connections (6 mm metric threads).
GST fusion proteins are easily purified from bacterial
lysates by affinity chromatography using immobilized
glutathione. GST fusion proteins are captured by the
affinity medium and impurities are simply removed by
washing. Fusion proteins are eluted under mild, nondenaturing conditions using reduced glutathione. The
purification process preserves protein antigenicity and
function. Three GST purification modules are available that use Glutathione Sepharose 4B in gravity
flow-, centrifugation-, or vacuum-based methods.
GSTrap affinity columns present a fourth option for
fast and easy purification of GST fusion proteins in a
convenient format. GSTrap columns are prepacked,
Life Science News 4, 2000 Amersham Biosciences
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Glutathione Sepharose Fast Flow
Easy operation
Glutathione Sepharose 4 Fast Flow, a high-capacity
affinity media with excellent flow properties, is the
base matrix for GSTrap 1 ml and 5 ml columns. The
glutathione ligand is coupled via a 10-carbon linker to
highly cross-linked 4% agarose. The coupling is
optimized to give a high binding capacity for GST
fusion proteins and other glutathione binding proteins. The total binding capacity is approximately
10 mg recombinant GST/ml gel. The dynamic binding
capacity will vary depending on flow rate and sample
properties. Glutathione Sepharose 4 Fast Flow is
available in lab packs for scale-up. Table 1 lists the
main characteristics of GSTrap and Glutathione
Sepharose 4 Fast Flow.
GSTrap columns are quick and easy to use. Instructions and connectors (M6 and luerlock) are included.
The columns can be used simply with a syringe for
loading, washing, and elution. Alternatively,
columns can be used with a pump or liquid chromatography system. The use of GSTrap columns
with ÄKTAexplorer 10 is highlighted below.
Purification of a GST fusion protein
An E. coli culture expressing a GST fusion protein was
harvested, washed and resuspended (1 g/10 ml) in
phosphate buffered saline (PBS, pH 7.3) supplemented
with 1 mM PMSF, 10 mM DTT, 100 mM MgCl2,
0.5 mg/ml lysozyme, and 1.7 U/ml DNase. The cell
suspension was incubated for 1 h at +4 ˚C with slow
magnetic stirring. The suspension was then frozen and
thawed to lyse the cells. The lysate was incubated at
+ 4 ˚C for an additional 30 min to cleave chromosomal
DNA and reduce viscosity. Cell debris was removed by
centrifugation at 47 000 × g, + 4 ˚C for 20 min. The
supernatant was filtered (0.2 µm filter) before applying to GSTrap.
Table 1.
Main characteristics of GSTrap and Glutathione Sepharose 4 Fast Flow.
Column dimensions i.d. × h
GSTrap 1 ml
0.7 × 2.5 cm
GSTrap 5 ml
1.6 × 2.5 cm
Column volumes
1 ml and 5 ml
Ligand
glutathione and 10-carbon linker arm
Ligand concentration
120–320 µmol glutathione/ml gel
Binding capacity
10 mg recombinant glutathione
S-transferase/ml gel (GST, Mw: 26 kDa)
GSTrap columns were equilibrated with 4 column
volumes (CV) PBS (pH 7.3) and the lysates were
applied (8 ml on GSTrap 1 ml and 40 ml on GSTrap
5 ml). The columns were washed with 10 CV PBS and
eluted using 5 CV 50 mM TrisCl (pH 8.0), 10 mM
reduced glutathione (Fig 2). The runs were completed
in 25 min using ÄKTAexplorer 10. Analysis by SDSPAGE indicated the isolation of highly pure GST
fusion protein (Fig 3). Fusion protein yields were
2.7 mg from GSTrap 1 ml and 13.4 mg from
GSTrap 5 ml.
11 mg GST fusion protein/ml gel Mw:
43 kDa (GSTrap 1 ml at 1 ml/min)
Mean particle size
90 µm
Bead structure
highly cross-linked 4% agarose
Maximum back pressure
GSTrap
0.3 MPa, 3 bar
Glutathione Sepharose 4
Fast Flow
0.1 MPa, 1 bar (packed in XK column.
May vary if used in other columns)
Maximum flow rates
GSTrap 1 ml
4 ml/min
GSTrap 5 ml
15 ml/min
On-column proteolytic cleavage
Glutathione Sepharose 4
Fast Flow
450 cm/h, (15 ml/min using XK 16/20
column, run at room temp. with
aqueous buffer)
Although many applications do not require the
removal of the GST moiety, it may be necessary when
the presence of an affinity tag raises concerns about
the physical or structural properties of the protein of
interest (e.g. crystallographic studies). To address this
need, all pGEX vectors include sequences between the
GST gene and the multiple cloning site that encode
protease recognition sites (thrombin, factor Xa, or
PreScission™ Protease). Treatment with the appropriate protease cleaves the GST moiety from the protein
of interest.
Recommended flow rates
Sample loading:
GSTrap 1 ml
0.2–1 ml/min
GSTrap 5 ml
1–5 ml/min
Glutathione Sepharose 4
Fast Flow
< 100 cm/h (< 3 ml/min using
XK 16/20 column)
Wash and elution:
GSTrap 1 ml
1–2 ml/min
GSTrap 5 ml
5–10 ml/min (5 ml)
Glutathione Sepharose 4
Fast Flow
100–300 cm/h (3–10 ml/min using
XK 16/20 column)
Chemical stability
All commonly used aqueous buffers,
e.g. 1 M acetate pH 4.0 and 6 M
guanidine hydrochloride for 1 hour
at room temperature
pH stability
pH 6–9
Storage temperature
+ 4–8 ˚C
Storage buffer
20% ethanol
Proteolytic cleavage can be performed in solution after
elution or while the fusion protein is bound to an
affinity medium (2), such as GSTrap. Cleaving while
bound to GSTrap eliminates the extra step of separating the released protein from GST because the GST
moiety remains bound to Glutathione Sepharose 4
Fast Flow.
Life Science News 4, 2000 Amersham Biosciences
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GSTrap 1 ml
8 ml cytoplasmic extract from E. coli
expressing a GST fusion protein
PBS, pH 7.3
50 mM TrisCl (pH 8.0) with
10 mM reduced glutathione
1 ml/min
Flow rate:
Chromatographic
procedure:
4 CV binding buffer, 8 ml sample,
10 CV binding buffer, 5 CV elution buffer,
5 CV binding buffer (CV = column volume)
Instrumentation: ÄKTAexplorer 10
A280
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Mr
(× 103)
%
elution
buffer
elution
buffer
3.5
94 –
3.0
100
2.5
67 –
2.7 mg
pure GST
fusion
protein
wash
2.0
80
43 –
60
1.5
30 –
40
1.0
20.1 –
20
0.5
14.4 –
0
0
5.0
5.0
B.
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thrombin solution
T
GST after thrombin
Binding buffer:
Elution buffer:
A
target after thrombin
Column:
Sample:
V
GSTrap 5 ml
O
GSTrap 1 ml
A.
N
lysate
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Column:
Sample:
Binding buffer:
Elution buffer:
10.0
10.0
15.0
15.0
ml
min
20.0
20.0
GSTrap 5 ml
40 ml cytoplasmic extract from E. coli
expressing a GST fusion protein
PBS, pH 7.3
50 mM TriHCl (pH 8.0) with 10 mM
reduced glutathione
5 ml/min
Fig 3. SDS-PAGE analysis of a GST fusion protein purified with
GSTrap affinity columns. A cytoplasmic extract from an E. coli culture
expressing a GST fusion protein was loaded onto GSTrap 1 ml and
GSTrap 5 ml columns (8 ml and 40 ml of lysate, respectively). Fusion
protein was eluted using 10 mM reduced glutathione (lanes marked
“GSTrap 1 ml” and “GSTrap 5 ml”). On-column cleavage with thrombin (20 U/ml) was performed with GSTrap 1 ml loaded with fusion
protein. The GST-free target protein was eluted with phosphatebuffered saline (lane marked “target after thrombin”) and the GST
moiety was eluted with 10 mM glutathione (lane marked “GST after
thrombin”). M = LMW Marker Kit (17-0446-01), reduced.
Flow rate:
Chromatographic
procedure:
4 CV binding buffer, 40 ml sample,
10 CV binding buffer, 5 CV elution buffer,
5 CV binding buffer (CV = column volume)
Instrumentation: ÄKTAexplorer 10
%
elution
buffer
A280
elution
buffer
3.5
3.0
Results from this technique are shown in Figures 3 and
4. A GST fusion protein containing the recognition
sequence for thrombin was applied to GSTrap 1 ml.
After washing, the column was filled by syringe with
1 ml thrombin solution (20 U/ml in PBS), prepared
according to the manufacturer’s protocol (Amersham
Biosciences, Fig 4A). The column was sealed
using the supplied connectors and left for 16 h at room
temperature. After incubation, the target protein
minus the GST moiety was eluted using PBS, and the
bound GST was eluted using elution buffer (Fig 4B).
The cleavage reaction yield was 100%. Intact GST
fusion protein was not detected in the glutathione
eluate (Fig 3).
100
wash
2.5
13.4 mg
pure GST
fusion
protein
2.0
80
60
1.5
40
1.0
20
0.5
0
0
20
4
40
8
60
12
80
16
100
20
ml
min
Fig 2. Purification of a GST fusion protein on GSTrap 1 ml (A) and
GSTrap 5 ml (B).
Life Science News 4, 2000 Amersham Biosciences
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Column:
Sample:
Binding buffer:
Elution buffer:
Flow rate:
Chromatographic
procedure:
Instrumentation:
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Summary
GSTrap 1 ml
10 ml cytoplasmic extract from
E. coli expressing a GST fusion protein
PBS, pH 7.3
50 mM TrisCl (pH 8.0) with
10 mM reduced glutathione
1 ml/min
GSTrap combines the versatility of HiTrap columns
with the high capacity and excellent flow properties of
Glutathione Sepharose 4 Fast Flow to provide a fast
and convenient method for purification of GST fusion
proteins. GSTrap columns are directly compatible
with existing purification protocols for GST fusion
proteins, including on-column proteolytic cleavage
methods. Purifications can be scaled-up easily by
connecting multiple columns in series, or larger
columns can be prepared using Glutathione Sepharose
4 Fast Flow, which is available in a lab pack size.
4 CV binding buffer, 10 ml sample,
10 CV binding buffer, filling column with
1 ml thrombin solution using a syringe
(CV = column volume)
ÄKTAexplorer 10
A280
3.5
3.0
N
wash
2.5
References
1. Smith, D.B. and Johnson, K.S., Gene 67, 31–40 (1988).
2. Gearing, D.P. et al., Bio/Technology 7, 1157–1161
(1989).
incubation
16 h
room temp.
2.0
1.5
1.0
0.5
0
5.0
B.
Column:
Sample:
Binding buffer:
Elution buffer:
Flow rate:
Chromatographic
procedure:
Instrumentation:
10.0
15.0
GSTrap 1 ml
column after 16 h incubation
with thrombin
PBS, pH 7.3
50 mM TrisCl (pH 8.0) with
10 mM reduced glutathione
1 ml/min
8 CV binding buffer (elution of
cleaved target protein), 5 CV elution
buffer (elution of free GST and
non-cleaved GST-fusion protein),
5 CV binding buffer
(CV = column volume)
ÄKTAexplorer 10
A280
3.5
3.0
100
2.5
GSTrap
2 × 1 ml
17-5130-02
GSTrap
5 × 1 ml
17-5130-01
GSTrap
1 × 5 ml
17-5131-01
Glutathione Sepharose 4 Fast Flow
25 ml*
17-5132-01
* Larger quantities are available. Please contact Amersham
Biosciences for more information.
%
elution
buffer
80
2.0
free
GST
1.5
1.0
ORDERING INFORMATION
min
60
target
protein
40
20
0.5
0
0
2.0
4.0
6.0
8.0
10.0
12.0 min
Fig 4. On-column thrombin cleavage of a GST fusion protein.
A. Equilibration, sample application, and washing of a GST fusion
protein on GSTrap 1 ml was performed using ÄKTAexplorer 10.
After washing, the column was filled by syringe with 1 ml thrombin
solution (20 U/ml PBS) and incubated for 16 h at room temperature.
B. GST-free target protein was eluted using PBS. GST was eluted
using 10 mM reduced glutathione. The GST-free target protein
fraction also contains a small amount of thrombin (not detectable
on SDS-PAGE, see Fig 3).
© Amersham Biosciences Ltd. 2000—All rights reserved.
Life Science News 4, 2000 Amersham Biosciences
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