Effect of flow rate, molecular weight of (His) -tagged proteins, and ...

Innovations Forum: Performance of HisTrap HP
Effect of flow rate, molecular weight of
(His) 6 -tagged proteins, and expression
system on the performance of
HisTrap HP columns
K. Öberg*, L. C. Andersson*, J. Lundqvist*, L. Anderson † , and H. Stålbrand †
*Amersham Biosciences AB, Uppsala, Sweden
† Department of Biochemistry, Lund University, Lund, Sweden
(His) 6 -tagged proteins were used in studies to evaluate the purification performance
of HisTrap HP columns. (His) 6 -tagged maltose-binding protein (MBP-[His] 6 , M r ~43 000)
expressed in E. coli was purified on HisTrap HP 1 ml columns at flow rates of 1, 2, or 4 ml/min.
The yield of MBP-(His) 6 was lower at the higher flow rates, but still satisfactory. Sample
purity remained high and unaffected. A (His) 6 -tagged mannanase (M r ~100 000) and
a hydrolase (M r ~34 000), were expressed in E. coli and P. pastoris, respectively. The
(His) 6 -mannanase was purified using a two-step protocol with a HisTrap HP column
followed by gel filtration on Superdex 200 10/300 GL; (His) 6 -hydrolase was purified
using a one-step IMAC protocol on a HisTrap HP column. High yield and purity of these
(His) 6 -tagged enzymes were observed. The results of this study show that the performance
of Ni Sepharose High Performance, the prepacked medium in HisTrap HP columns, was
not limited by flow rate, molecular weight of the three (His) 6 -tagged proteins used, or
the two expression systems employed.
Introduction
The (His) 6 tag is the most used affinity tag due to its small size,
strong metal ion binding, and ability to bind under denaturing,
as well as native conditions (1, 2). Immobilized metal ion affinity
chromatography (IMAC) with nickel-charged media is a welldocumented
method for purifying histidine-tagged proteins ✧ (3).
HisTrap HP columns, prepacked with Ni Sepharose High Performance,
are effective and convenient tools for this application. The medium,
supplied precharged with Ni 2+ ions, strongly binds His-tagged
proteins, which are then easily eluted from the column.
The aim of this study was to evaluate the performance of prepacked
HisTrap HP columns 1) at different flow rates; 2) using (His) 6
-tagged
proteins of diverse molecular weight; 3) in one-step or two-step
purification of (His) 6
-tagged proteins expressed in different hosts.
High yield and purity at increased flow rate
C-terminally (His) 6
-tagged maltose binding protein, MBP-(His) 6
, was
subcloned and expressed in E. coli. Prior to cell lysis, PMSF was added
to a final concentration of 1 mM to prevent proteolysis. Sodium
chloride and imidazole were added to the cell lysate to give final
concentrations of 500 mM and 35 mM, respectively; the pH was
adjusted to 7.4. The cell lysate was centrifuged and filtered prior to
IMAC. Extracts with MBP-(His) 6 were loaded onto equilibrated 1-ml
HisTrap HP columns at flow rates of 1, 2, or 4 ml/min. All steps in the
purification procedure i.e. equilibration, sample application, and wash
were performed at these flow rates. The imidazole concentration
during equilibration, sample application, and wash was 35 mM, which
reduced the amount of contaminating E. coli proteins and allowed
selective binding of MBP-(His) 6
. One-step elution was performed
with buffer containing 500 mM imidazole and 1-ml fractions
were collected. The concentration and yield of MBP-(His) 6
were
estimated by measurement at 280 nm. Sample purity was
analyzed by SDS polyacrylamide gel electrophoresis using
ExcelGel SDS Gradient 8–18.
A decrease in yield of MBP-(His) 6 was observed in runs at the higher
flow rates of 2 and 4 ml/min (Fig 1). This can, in many cases, be
regarded as an acceptable loss. The loss at high flow rates might be
minimized by washing at a lower concentration of imidazole than the
35 mM used here. MBP-(His) 6
purity was unaffected by changes in
flow rate as indicated in Figure 2.
✧ See licensing information on page 2.
Life Science News 18, 2004 Amersham Biosciences 3

Innovations Forum: Performance of HisTrap HP
3000
2000
1000
Fig 1. Purification of MBP-(His) 6
with different flow rates on HisTrap HP columns. Yields
for 1, 2, and 4 ml/min flow rates were 11, 9, and 8 mg MBP-(His) 6
, respectively.
M r
97 000
66 000
45 000
30 000
20 100
14 400
Column:
HisTrap HP 1 ml
Sample: E. coli extract containing MBP-(His) 6
Sample load: 8 ml
Binding buffer: 20 mM sodium phosphate, 500 mM sodium chloride,
35 mM imidazole, pH 7.4
Elution buffer: 20 mM sodium phosphate, 500 mM sodium chloride,
500 mM imidazole, pH 7.4
Elution:
One-step elution with elution buffer
Flow rates: 1, 2, or 4 ml/min
System: ÄKTAexplorer 10
A 280
(mAU)
0
0.0
LMW
E. coli extract,
dil. 1:20
8 mg
9 mg
10.0 20.0 30.0 40.0
1 ml/min
48 min
11 mg yield
1 2 3
2 ml/min
24 min
9 mg yield
1 2 3
1 ml/min
2 ml/min
4 ml/min
11 mg
4 ml/min
12 min
8 mg yield
1 2 3
Fig 2. Purity analysis of eluted MBP-(His) 6 as determined by SDS-PAGE on Multiphor II.
The gel was Coomassie stained. Lanes 1–3 shown for each flow rate.
Lane 1: flowthrough diluted 1:10 with sample buffer; lane 2: wash, undiluted;
lane 3: eluted MBP-(His) 6 pool diluted 1:5.
Two-step purification of a high molecular weight
(His) 6 -tagged protein expressed in E. coli
A (His) 6
-tagged mannanase (Man 26A from Cellulomonas fimi)
was expressed in E. coli. Sample preparation was performed according
to the procedures described for MBP-(His) 6
. Initial purification was
min
performed using Ni Sepharose High Performance. The buffer used
for equilibration, sample application, and wash contained 30 mM
imidazole, which optimized recovery of this particular protein.
Extract with (His) 6 -Man 26A was loaded onto an equilibrated
HisTrap HP column at a flow rate of 1 ml/min. Elution was performed
using a linear gradient of elution buffer and 1-ml fractions of eluted
(His) 6 -Man 26A were collected, analyzed, and pooled. One ml of the
concentrated pool was loaded onto a Superdex 200 10/300 GL
column for gel filtration. Sample purity was analyzed by SDS-PAGE
as described previously.
The results of this purification are shown in Figure 3. Enzymatically
active, pure (His) 6
-Man 26A was obtained using the two-step
protocol described.
One-step purification of a (His) 6
-tagged
protein expressed in P. pastoris
A (His) 6 -tagged hydrolase (putative hydrolase from Saccharomyces
cerevisiae) was expressed in Pichia pastoris. The methods used
for IMAC of the clarified cell lysate were as described for
(His) 6 -mannanase except that 85 mM imidazole was used
during equilibration, sample application, and wash. Sample
purity was analyzed by SDS-PAGE as described previously.
The one-step IMAC purification resulted in the recovery of pure
(His) 6 -hydrolase (Figs 4–5).
Conclusions
The excellent binding properties of Ni Sepharose High Performance
allow high flow rate purifications of MBP-(His) 6 with an acceptable
loss of yield and unaffected purity. Moreover, effective purification of
(His) 6 -tagged proteins with different molecular weights obtained from
different expression systems was possible using HisTrap HP columns.
References
1. Gaberc-Porekar, V. and Menart, V. Perspectives of immobilized-metal
affinity chromatography. J. Biochem. Biophys. Methods 49, 335–360 (2001).
2. Ueda, E. K. M. et al. Current and prospective applications of metal ion
protein binding. J. Chromatogr A 988, 1–23 (2003).
3. The Recombinant Protein Handbook: Protein Amplification and Simple
Purification, Amersham Biosciences, 18-1142-75 AB (2001).
Ordering Information
HisTrap HP (5 × 1 ml) 17-5247-01
HisTrap HP (100 × 1 ml*) 17-5247-05
HisTrap HP (1 × 5 ml) 17-5248-01
HisTrap HP (5 × 5 ml) 17-5248-02
HisTrap HP (100 × 5 ml*) 17-5248-05
HisTrap HP Kit 17-5249-01
(includes 3 × 1 ml columns plus binding and elution buffers)
Ni Sepharose High Performance (25 ml) 17-5268-01
Ni Sepharose High Performance (100 ml) 17-5268-02
* Pack size available by special order.
To shop online, go to www.amershambiosciences.com
To obtain brochures on the products, please visit www.lsn-online.com/more-info.
4
Life Science News 18, 2004 Amersham Biosciences

Innovations Forum: Performance of HisTrap HP
IMAC conditions
Column:
HisTrap HP 1 ml
Sample:
E. coli extract containing (His) 6
-Man 26A
Sample load: 10 ml
Binding buffer: 20 mM sodium phosphate, 500 mM sodium
chloride, 30 mM imidazole, pH 7.4
Elution buffer: 20 mM sodium phosphate, 500 mM sodium
chloride, 500 mM imidazole, pH 7.4
Elution:
25 ml linear gradient 30–300 mM imidazole
Flow rate:
1 ml/min
System: ÄKTAexplorer 10
Gel filtration conditions
Column:
Superdex 200 10/300 GL
Buffer:
20 mM sodium phosphate, 500 mM sodium
chloride, pH 7.4
Flow rate:
0.5 ml/min
System: ÄKTAexplorer 10
A 280
(mAU)
A 280
(mAU)
80.0
60.0
40.0
20.0
(B)
0.0
0.0 10.0 20.0 30.0 ml
LMW
E. coli extract
IMAC flowthrough
Early IMAC fraction
IMAC eluted pool
Gel filtration pool
Fig 3. Two-step
purification of (His) 6 -
Man 26A expressed in
E. coli. (A) First-step IMAC
using HisTrap HP. (B) The
fractions between the
black lines were pooled
and further purified by gel
filtration on Superdex 200
10/300 GL. (C) Purity
analysis and confirmation
of the apparent molecular
weight of (His) 6 -Man 26A.
The purified (His) 6 -Man
26A is indicated by the
arrow. Note: Previous
results have indicated that
the contaminants present
after IMAC include various
truncated, His-tagged
forms of the mannanase
(data not shown).
(A)
400
M r
97 000
300
66 000
45 000
200
30 000
100
20 100
14 400
0
0.0 10.0 20.0 30.0 40.0 50.0 ml
(C)
A 280
(mAU)
2500
2000
1500
P. pastoris extract
Flowthrough
Final pool
LMW
Fig 5. SDS-PAGE results of
the one-step purification
of (His) 6
-hydrolase. (His) 6
-
hydrolase in the extract is
indicated by the arrow.
1000
500
0
0 20 40 60 80 100 ml
M r
97 000
66 000
45 000
Fig 4. Purification of (His) 6
-hydrolase expressed in P. pastoris on HisTrap HP 1 ml.
Running conditions were as shown in Figure 1 with the following exceptions: 50 ml of
extract was applied; the binding buffer and extract contained 85 mM imidazole; the linear
gradient employed was 25 ml 85–300 mM imidazole; and the flow rate was 1 ml/min.
30 000
20 100
14 400
Life Science News 18, 2004 Amersham Biosciences 5