11254 - Discovery Matters Issue 2 4/10/05 2:30 pm Page 24
INNOVATIONS FORUM
Protein purification and production
New gravity-flow column gives faster and simpler
purification of histidine-tagged proteins
A. Bergh, M. Carlsson, A. Karlsson, and H. Lindgren
GE Healthcare, Uppsala, Sweden
Histidine-tagged proteins* can be quickly and simply
purified on His GraviTrap columns prepacked with Ni
Sepharose 6 Fast Flow without the need for a pump or
purification system. The protein binding capacity
(approx. 40 mg/column) is among the highest available
on the market, and separations can be completed in
as little as 30 min. Large volumes of clarified or
unclarified samples can easily be applied and the
purified protein can be eluted in a small volume
resulting in a highly concentrated target protein.
Introduction
Immobilized metal ion affinity chromatography (IMAC) is a
widely used and highly effective technique that exploits the
affinity of histidine-tagged proteins to metal ions attached
to chelating separation media, such as Ni Sepharose 6 Fast
Flow. His GraviTrap is a single-use column prepacked with
Ni Sepharose 6 Fast Flow. His GraviTrap provides high
protein binding capacity, short purification times, and simple
operation.
The purification of histidine-tagged proteins on His
GraviTrap can be divided into four stages: equilibration,
sample application, washing, and elution (Fig. 1).
Purifications can be performed either under native or
denaturing conditions, and a number of additives can be
used. In addition, new accessory products further increase
speed and convenience.
His GraviTrap and accessories
His GraviTrap contains 1 ml of Ni Sepharose 6 Fast Flow,
which delivers a protein binding capacity of approximately
40 mg histidine-tagged protein/column. The columns are
made of bio-inert polypropylene, and special frits protect
the medium from running dry during use. The column
packaging can be converted into a stand (Workmate), and
the plastic tray in this package can be used to collect
liquid waste.
The optional LabMate reservoir increases the loading
capacity from 10 ml to approximately 35 ml. Large volumes
can thus be applied in one go without having to constantly
attend the column to add more sample.
The new His Buffer Kit delivers added convenience by
providing phosphate buffer concentrates and highly pure
2 M imidazole stock solutions. This kit eliminates time-
consuming manual buffer preparation, thereby promoting
optimal convenience, performance, and reproducibility.
Faster purifications under a wide range of
conditions
The performance of His GraviTrap was compared with
Ni-NTA Superflow™ gravity-flow column (Qiagen Inc.) during
the purification of histidine-tagged green fluorescent protein
(GFP-[His]6; Mr 28 000) from an E. coli lysate. The imidazole
concentration in the sample and binding buffers was 45 mM
for His GraviTrap and 10 mM for Ni-NTA Superflow (the latter
according to manufacturer’s recommendations).
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The recovery was greater than 98% in the first 3 ml eluate
from His GraviTrap, and approximately 80% from the other
column. For complete elution from Ni-NTA Superflow, a total
volume of 6 to 9 ml was needed, and the total purification
time was close to 100 min. The total purification time was
20 min when His GraviTrap was used (Fig 2A). The purity of
the eluates was similar as determined by SDS-PAGE (data
not shown).
His GraviTrap and Ni-NTA Superflow columns were also
compared under denaturing conditions. Histidine-tagged
Maltose Binding Protein (MBP-(His)6; Mr 43 000) in E. coli
BL-21 lysate containing 7 M urea was applied to the
columns. Elution was achieved by lowering the pH from pH
8.0 to pH 4.5. Although denaturing conditions increase the
purification time due to the higher viscosity of the samples
and buffers, total purification time was still approximately
four times faster on His GraviTrap (Fig 2B).
Complete details of these comparisons are
provided in data file 11-0036-90, available at
www.amershambiosciences.com/promo_dm205
His GraviTrap also enables rapid separation of large
proteins. To demonstrate this, His GraviTrap was used to
purify (His)10-TRX-P450 (Mr 133 200). E. coli JM109 cells
expressing (His)10-TRX-P450 were enzymatically and
mechanically lysed and a 20 ml clarified sample was loaded
onto His GraviTrap using LabMate. The whole purification
took just 25 min.
Results show that the purification yielded three major
protein bands when analyzed by SDS-PAGE (Fig 3A, lane 4).
Western blot analysis confirms that each of the three bands
contains a histidine-tag (Fig 3B). However, only a weak band
can be seen from the band at approximately Mr 15 000.
Some of the smaller proteins may have passed through the
membrane due to a prolonged blotting time. N-terminal
sequencing (data not shown) of the three bands confirms
that the low molecular weight bands are truncated forms of
the histidine-tagged target protein. To separate the full-
length protein from the truncated forms, a second
purification step, such as gel filtration, is recommended.
Summary
His GraviTrap columns provide a very fast and simple
method for purifying histidine-tagged proteins without the
need for a pump or purification system. The prepacked Ni
Sepharose 6 Fast Flow gives high protein binding capacity.
Proven protocols, application data, and new support
products help optimize purification results and simplify the
whole procedure.
To obtain data file 11-0036-90, visit
www.amershambiosciences.com/promo_dm205
Acknowledgements
GFP-(His)6 was provided by Dr. David Drew, Dept. of
Biochemistry and Biophysics, Stockholm University,
Stockholm, Sweden. MBP-(His)6 was provided by Pharmacia
Diagnostics, Uppsala, Sweden.
11254 - Discovery Matters Issue 2 4/10/05 2:30 pm Page 25

INNOVATIONS FORUM
Protein purification and production
A.

— (His)10-TRX-P450

Fig 1. Purifying histidine-tagged proteins with His GraviTrap is

a simple four-stage procedure.

B.

A.
— (His)10-TRX-P450

B.
Fig 2. Comparison of total purification time for His GraviTrap
and Ni-NTA Superflow gravity-flow column. (A) Under native
conditions, His GraviTrap was almost five-times faster. (B) Under
denaturing conditions, His GraviTrap was approximately four-
times faster.
Fig 3. SDS-PAGE and Western blot analysis of the purification of
(His)10-TRX-P450 on His GraviTrap. (His)10-TRX-P450 was purified
with His GraviTrap using a binding buffer of 20 mM sodium
phosphate, 500 mM NaCl, 40 mM imidazole, pH 7.4. The elution
buffer was the same except that 500 mM imidazole was used.
(A) SDS-PAGE of fractions from the purification of (His)10-TRX-P450
on His GraviTrap (electophoresis was performed using
PhastSystem™ electrophoresis sytem, PhastGel™ Gradient 10-15
gel, under reduced conditions with Coomassie™ Blue staining).
(B) Western blot using PhastTransfer™ Unit and Hybond ECL.
(Primary antibody: Anti-His. Secondary antibody: Anti-mouse
IgG-HRP). The E. coli JM109 lysate was diluted 1:20 for
electrophoresis. The amount of histidine-tagged protein detected
in the flowthrough is a result of column overloading. M = High-
Range Rainbow Molecular Weight Markers (RPN756). Negative
control = untransformed E. coli JM109.

Ordering information
Product Code number
His GraviTrap (10 x 1 ml) 11-0033-99
His GraviTrap Kit (includes 2 packs His GraviTrap and 1 pack His Buffer Kit) 28-4013-51
LabMate PD-10 Buffer Reservoir (10) 18-3216-03
His Buffer Kit (includes 2 x 100 ml phosphate buffer, 8x stock solution, pH 7.4; 1 x 100 ml 2 M imidazole, pH 7.4) 11-0034-00
High-Range Rainbow Molecular Weight Markers (250 µl) RPN756
Hybond ECL (20 x 20 cm, 10 sheets) RPN2020D
Anti-His Antibody (170 µl) 27-4710-01
Mouse IgG, HRP-Linked Whole Ab (from sheep) (100 µl) NA931-100UL
PhastSystem Separation-Control and Development Units 120 VAC 18-1018-23
PhastSystem Separation-Control and Development Units 220 VAC 18-1018-24
PhastTransfer Kit 18-1001-23
PhastGel Gradient 10–15 (10 gels) 17-0540-01
PhastGel Buffer Strips - SDS (10 pairs) 17-0516-01

* See licensing information on page 28.

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