I N N O V A T I O N S F O R U M

An efficient and rapid protein purification and

on-column cleavage strategy using GSTrap Columns
R. Knaust*, S. Eshaghi*, C. Dian*, S. Lundgren*, K. Anzai†, P. Nordlund*, and D. Birse*, ‡
* The Structural Biochemistry Unit, Department of Biochemistry, Stockholm University, Stockholm, Sweden
† Department of Biochemistry, College of Pharmacy, Nihon University, Chiba, Japan
‡ To whom correspondence should be addressed

This paper describes a flexible, efficient and rapid Protein expression

P protein purification scheme for the purification of The genes encoding for Pur-α and TLP40 protein were
R GST-fusion proteins using GSTrap™ columns and independently subcloned into pGEX-6P-1 and were
O PreScission™ Protease. The method can be used to transformed into E. coli BL21. The bacteria are grown
T
reproducibly isolate a variety of proteins of differing at 30 ˚C in LB medium supplemented with 100 µg/ml
E P function, structure, and chemical nature. This strategy carbenicillin in Tunair™ shaking flasks (Shelton Scien-
I U
N
is performed on two, 5 ml GSTrap columns connected tific). The cells were grown until OD600 ≈ 1.0 and then
R in series, yielding “native” proteins (cleaved fusion GST-fusion protein synthesis was induced with 1.0 mM
I
proteins) in high yield with a purity of 95–97%. IPTG (final concentration). Induced cultures were
F
I incubated for an additional 3–4 h. Cells were har-
Introduction vested by centrifugation (3000 × g for 30 minutes
C We’ve developed a purification strategy designed to
A at 4 ˚C). The pellet was resuspended and washed with
produce highly pure, “native” protein suitable for TB buffer (9.1 mM HEPES, 55 mM MgCl2, 15 mM
T
downstream biochemical assays and structural studies, CaCl2, 250 mM KCl, pH 6.7), centrifuged, and frozen
I
such as protein crystallographic analysis. The method at -80 ˚C.
O
described here utilizes the GST Gene Fusion System✧,
N
in particular, pGEX-6P vectors, Precission Protease, Cell lysis
and GSTrap affinity columns. The cell pellet was resuspended in PBS lysis buffer
pGEX-6P vectors contain sequences between the GST (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4,
gene and the multiple cloning site (MCS) that encode 1.8 mM KH2PO4, pH 7.4) supplemented with
an optimal recognition site for PreScission Protease. lysozyme (1 mg/ml), Complete Protease Inhibitor
PreScission Protease is a fusion of GST with human Cocktail (1 tablet, Roche Molecular Biochemicals),
rhinovirus 3C protease (1), which permits the simulta- 10 mM MgCl2, and DNAse I (10 U/ml). The cells
neous digestion of GST-fusion proteins and removal of were lysed by three freeze (-170 ˚C)/thaw (30 ˚C)
both GST and protease from the protein of interest. cycles. The lysate was centrifuged at 70 000 × g for
24 min at 4 ˚C. The clarified lysate was centrifuged at
GSTrap affinity columns are prepacked, ready-to-use 300 000 × g for 60 min at 4 ˚C to remove insoluble
1 ml and 5 ml HiTrap™ columns containing Glutathione proteins, cell fragments and membrane components.
Sepharose™ 4 Fast Flow. The columns are designed for
one-step purification of GST fusion proteins. GST-fusion protein binding
The following purification procedure was performed
The purification method was developed using proteins
using an ÄKTA™explorer 100 chromatography system
that had previously required complex multi-step purifi-
(Amersham Biosciences). The final supernatant
cation methods, which often yielded unsatisfactory
was applied to a 50 ml Superloop™ (Amersham
results. One protein used was Pur-α, which is a
Biosciences) and loaded onto two pre-
sequence-specific, single-stranded DNA and RNA
equilibrated GSTrap 5 ml columns connected in series.
binding protein that binds to purine-rich promoter
PBS was used as binding buffer. The flow rate for
regions with a consensus (GGN)n sequence (2). Another
loading was 1 ml/min, a rate found optimal for fusion
protein used was TLP40, which is a eukaryotic plant
protein binding. Loaded columns were washed with
protein involved in photosystem II regulation (3).
PBS until the baseline stabilized. Once the baseline
With the new method, these proteins were sub-cloned,
stabilized, the buffer was switched to PreScission
expressed, and purified yielding highly pure product.
buffer (50 mM Tris-HCl, 100 mM NaCl, 1 mM
✧
EDTA, 1 mM DTT adjusted to pH 8.0). Equilibration
See licensing information on page 3.

Life Science News 6, 2000 Amersham Biosciences

1
I N N O V A T I O N S F O R U M

with PreScission buffer was continued until both the The different stages of the purification process were
UV absorbance baseline and the conductivity baseline analysed by SDS-PAGE (Fig 2). The purity of the final,
stabilized, after which buffer flow was stopped. cleaved target protein was in the range of 95–97%.

On-column cleavage with PreScission Protease 1 2 3 4 5 6

Mr
For PreScission Protease digestion, 2 units enzyme/
100 µg of bound GST-fusion protein was used.
PreScission Protease was diluted in PreScission buffer 94 —
(9 ml total volume for two columns in series) and
67 —
manually injected into the columns at an increased
flow rate of 5–7 ml/min. This increased flow allows
for uniformly distributed enzyme throughout the 43 —
GSTrap columns. Following injection, the column was
closed, sealed and incubated for 12–16 h at 4 ˚C.

Column: 2 × 5 ml GSTrap in series 30 —

Sample: TLP40 (50 ml lysate from 2 l culture)
Buffer A: PBS, pH 7.4
Buffer B: PreScission buffer, pH 8.0
Buffer C: Reduced glutathione buffer, pH 8.0
System: ÄKTAexplorer 100
20 —
mAU %B
100
Buffer C

4000 eluted GST-tag 80 Fig 2. SDS-PAGE analysis of the steps in the purification strategy.
Reduced Glutathione buffer

Samples were separated on a 12% polyacrylamide gel. Lane 1, total

Absorbance (280 nm)

protein extract of non-induced culture; lane 2, total protein extract of

3000 cleaved TLP40 fraction 60 induced culture; lane 3, supernatant after 70 000 × g centrifugation
step; lane 4, supernatant after 300 000 × g centrifugation step; lane 5,
flow-through from GSTrap column; lane 6, “native” TLP40 cleavage
2000 40 product eluted after on-column digest with PreScission Protease.
PreScission Protease
Buffer B
Buffer A

1000 20
Conclusions
0
F2 23456789 12 14 16 18 20 22 2426
0
The described protein purification strategy is a simple
0 50 100 150 200 ml and rapid method for the isolation of soluble “native”
Fig 1. Purification and on-column cleavage of GST-TLP40 fusion pro- proteins. The protein yields are highly pure and suit-
tein using GSTrap and PreScission Protease.
able for downstream processes. In one of the above
test cases, the protein was crystallized, indicating the
highly pure and homogeneous state of the protein.
Elution of cleaved protein
Prior to elution, a 1 ml GSTrap column (pre-equilibrated References
with PreScission buffer) was connected downstream to 1. Walker, P. A. et al., Bio/technology 12, 601 (1994).
the GSTrap proteolytic cleavage columns. The 1 ml 2. Bergemann, A. D. and Johnson, E. M., Mol Cell Biol 12,
1257–1265 (1992).
column was placed in-line to detain any cleaved
3. Fulgosi, H. et al., EMBO J 17, 1577–1587 (1998).
material upon flow start-up. This configuration
minimized loss of cleaved product and allowed for
rapid baseline recalibration before peak elution. The
1 ml column also acted as a filter to capture any
released cleaved GST protein, uncleaved GST-fusion
protein and unbound PreScission Protease. ORDERING INFORMATION
GSTrap 2 × 1 ml 17-5130-02
Elution of cleaved protein occurred immediately upon GSTrap 5 × 1 ml 17-5130-01
flow start-up (Fig 1). After target protein elution, GSTrap 1 × 5 ml 17-5131-01
reduced glutathione (50 mM Tris-HCl and 10 mM PreScission Protease 500 units 27-0843-01

reduced glutathione adjusted to pH 8.0) was applied in pGEX-6P-1 25 µg 27-4597-01

pGEX-6P-2 25 µg 27-4598-01
a full one-step gradient (100%) to elute GST, unbound
pGEX-6P-3 25 µg 27-4599-01
GST-fusion protein and PreScission Protease (Fig 1).

Life Science News 6, 2000 Amersham Biosciences