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

Innovations Forum: Performance of HisTrap HP
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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.
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Life Science News 18, 2004 Amersham Biosciences