Simplified purification of secreted
histidine-tagged proteins
Marianne Carlsson
GE Healthcare Life Sciences
Outline
Introduction
Applications
Summary
2
WT North America
17/04/2012
Immobilized Metal Ion Affinity
Chromatography (IMAC)
Histidine-tag
Ligand
Matrix
Me2+
Protein
Advantages of IMAC
Histidine-tag
Small with low charge
Stable
Compatible with many chemicals
Compatible with denaturing conditions
Chromatography medium
High binding capacity
Controllable selectivity
Mild elution conditions
Matrix
4
WT North America
A challenge in IMAC purification
Secretion into cultivation media for eukaryotic cells
• Interfering substances: Chelators (e.g. EDTA), histidine, arginine, etc.
• Low target protein concentration  Large sample volumes (> 5 L)
Issue:
 Stripping of immobilized Me2+ from conventional
IMAC resins
 No or low binding of histidine-tagged protein
5
Conventional IMAC
Protein secreted into CHO cell culture medium
Resin:
Sample:
System:
Ni Sepharose™ 6 Fast Flow (FF), 1 ml (column Ø=5 mm)
250 ml of mPAI-1-(his)8 secreted into GIBCO™ CD CHO medium, pH 7.0
ÄKTA™ avant 25
Mr x 103
97.0
66.0
45.0
30.0
20.1
14.4
1
Equilibration buffer:
Wash buffer:
Elution buffer:
2
3
4
5
20 mM sodium phosphate, 500 mM NaCl, pH 7.4
20 mM sodium phosphate, 500 mM NaCl, 10 mM imidazole, pH 7.4
20 mM sodium phosphate, 500 mM NaCl, 500 mM imidazole, pH 7.4
6
Remedy
Common workflow
Cell cultivation
Removal of cells
Diafiltration
Final concentration and
clarification
IMAC with conventional
Me2+ resin
7
Remedy
Common workflow
Cell cultivation
Removal of cells
Diafiltration
Final concentration and
clarification
IMAC with conventional
Me2+ resin
8
Remedy
Common workflow
Simplified workflow
Cell cultivation
Cell cultivation
Removal of cells
Removal of cells
Diafiltration
Final concentration and
clarification
IMAC with conventional
Me2+ resin
IMAC with
Ni Sepharose™ excel
HisTrap™ excel
His Mag Sepharose excel
Conventional IMAC
Protein secreted into CHO cell culture medium
Resin:
Sample:
System:
Ni Sepharose™ 6 Fast Flow (FF), 1 ml (column Ø=5 mm)
250 ml of mPAI-1-(his)8 secreted into GIBCO™ CD CHO medium, pH 7.0
ÄKTA™ avant 25
Mr x 103
97.0
66.0
45.0
30.0
20.1
14.4
1
Equilibration buffer:
Wash buffer:
Elution buffer:
2
3
4
5
20 mM sodium phosphate, 500 mM NaCl, pH 7.4
20 mM sodium phosphate, 500 mM NaCl, 10 mM imidazole, pH 7.4
20 mM sodium phosphate, 500 mM NaCl, 500 mM imidazole, pH 7.4
10
Simplified IMAC
Protein secreted into CHO cell culture medium
Resins:
Sample:
System:
Ni Sepharose™ excel (blue) and Ni Sepharose 6 FF (green), 1 ml (column Ø=5 mm)
250 ml of mPAI-1-(his)8 secreted into GIBCO™ CD CHO medium, pH 7.0
ÄKTA™ avant 25
Ni Sepharose
6 FF
excel
Mr x 103
97.0
66.0
45.0
30.0
20.1
14.4
1
2
3
4
5
6
7
8
Equilibration buffer: 20 mM sodium phosphate, 500 mM NaCl, 0 mM (excel) or 20 mM (FF) imidazole, pH 7.4
Wash buffer:
20 mM sodium phosphate, 500 mM NaCl, 10 mM (excel) or 20 mM (FF) imidazole, pH 7.4
Elution buffer:
20 mM sodium phosphate, 500 mM NaCl, 500 mM imidazole, pH 7.4
11
Nickel leakage (%)
Nickel leakage
45
40
35
30
25
20
15
10
5
0
Ni Sepharose™ excel
Ni Sepharose 6 FF
EX-CELL™ 420
(SIGMA-ALDRICH)
GIBCO™ CD CHO
(Invitrogen)
TC 100
(PAA)
Cultivation medium
Test:
Analysis:
1 ml of each resin, incubated in 5 ml cultivation media for 24h at room temperature.
Nickel leakage was determined by elemental analysis.
12
Compatibility with EDTA
Column:
Sample:
System:
HisTrap™ excel 1 ml
250 ml of proCPU-(his)8 secreted into GIBCO™ Sf-900 II insect cell medium, pH 6.8, later
supplemented with 0 mM (dark blue), 2 mM (purple), or 10 mM (pale blue) EDTA
ÄKTA™ avant 25
Eluates
Mr x 103
97.0
66.0
45.0
30.0
20.1
14.4
1
Equilibration buffer:
Wash buffer:
Elution buffer:
2
3
4
20 mM sodium phosphate, 500 mM NaCl, pH 7.4
20 mM sodium phosphate, 500 mM NaCl, 15 mM imidazole, pH 7.4
20 mM sodium phosphate, 500 mM NaCl, 500 mM imidazole, pH 7.4
13
Outline
Introduction
Applications
Summary
14
Protein secreted into insect cell
culture medium
Columns:
Sample:
Sample volumes:
System:
HisTrap™ excel 5 ml (blue) and HisTrap FF crude 5 ml (purple)
(his)6-HA secreted into GIBCO™ Sf-900 II SFM insect cell culture medium, pH 6.6
1020 ml and 1012 ml, respectively
ÄKTA™
M SW
HisTrap excel 5 ml : Yield 8.9 mg of target protein
HisTrap FF crude 5 ml: Yield too low to be quantified
excel
E
M: Molecular Weight Standard
S: Sample
W: Wash
E: Elution fractions
Data from Dr. Linda Lua et al., UQ Protein Expression Facility, the University of Queensland, Brisbane, Australia.
FF
SW E
Effect of sample pH on yield
Ni Sepharose™ excel, 0.25 ml (column Ø=5 mm)
35 ml of 0.1 mg/ml MBP-(his)6 added to SAFC EX-CELL™ 405 culture medium, pH 5 to 8
ÄKTA™ avant 25
Eluted peak area (mAu*ml)
Resin:
Sample:
System:
5000
MBP-(his)6
in EX-CELL
4000
3000
MBP-(his)6
in control
buffer
2000
1000
0
4,5
5,5
6,5
pH
7,5
8,5
 Yields are equal at sample pH values ranging from 6.0-7.5
16
Impact of imidazole on purity and yield
Resin:
Sample:
His Mag Sepharose™ excel, 200 µl 10% medium slurry
10 ml PRCP-(his)9 secreted into SAFC EX-CELL™ 405 culture medium, pH 6.9
Wash
Eluates
Wash
Eluates
Wash
Eluates
Wash
Eluates
97.0
66.0
45.0
30.0
20.1
14.4
0 mM imidazole
10 mM imidazole
30 mM imidazole
50 mM imidazole
100
80
80
60
60
40
40
20
20
0
0
Purity (%)
Mr x 103
Relative yield (%)
Imidazole conc was varied in the wash buffer: 0 mM (red), 10 mM (purple), 30 mM (green) and 50 mM (blue)
0 10 20 30 40 50
Wash buffer imidazole conc
(mM)
Higher imidazole concentration  higher purity & lower yield
17
50-fold scale-up
His Mag Sepharose™ excel  HisTrap™ excel column
Resin/column:
His Mag Sepharose excel, 200 µl 10% medium slurry (blue) scaled up to HisTrap
excel, 1 ml column (purple)
Sample:
10 and 500 ml PRCP-(his)9 secreted into SAFC EX-CELL™ 405 culture medium, pH 6.9
30 mM imidazole was used in wash buffer for scale-up experiment
Mr x 103 LMW
His Mag HisTrap
S 200 µl 1 ml
97.0
66.0
45.0
30.0
20.1
14.4
LMW: LMW-SDS Marker Kit
S: Sample
18
Outline
Introduction
Applications
Summary
17/04/2012
Summary
IMAC purification of proteins secreted into eukaryotic cell
culture media is simplified using Nickel Sepharose™ excel
• Exceptionally strong binding of Ni2+
• Minimal sample pretreatment
• No diafiltration or other buffer-exchange procedures needed
• Broad pH range
• Flexibility
Acknowledgements
• Dr. Linda Lua and members of UQ Protein
Expression Facility, the University of
Queensland, Brisbane, Australia
• Dr. Wolfgang Knecht, CVGI iMed Bioscience
and Dr. Paul Wan, Discovery Sciences,
AstraZeneca R&D, Mölndal, Sweden
21
Thank you!
ÄKTA, HisTrap, and Sepharose are trademarks of GE Healthcare companies. GE and GE Monogram are trademarks of General Electric
Company.
GIBCO is a trademark of Life Technologies Corporation. EX-CELL is a trademark of Sigma-Aldrich Co.
Purification and preparation of fusion proteins and affinity peptides comprising at least two adjacent histidine residues may require a
license under US patent numbers 5,284,933 and 5,310,663, and equivalent patents and patent applications in other countries assignee:
Hoffman La Roche, Inc.).
Ni Sepharose 6 Fast Flow products are sold under a license from Sigma-Aldrich under patent number EP 1277616 (Metal chelating
compositions) and equivalent patents and patent applications in other countries.
All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them. A copy
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