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Developing an efficient protein purification scheme. Developing an efficient protein purification scheme. Introduction Three phase strategy Combining techniques Purity requirements Characteristics of the target protein and contaminants Examples Summary and shortcuts. - PowerPoint PPT Presentation
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2Strategy/AC/1998/JB/Å. Danielsson
Introduction Three phase strategy Combining techniques Purity requirements Characteristics of the target protein and
contaminants Examples Summary and shortcuts
Developing an efficient protein purification schemeDeveloping an efficient protein purification scheme
3Strategy/AC/1998/JB/Å. Danielsson
Protein Purification - AimsProtein Purification - Aims
Sufficient purity and quantity Maintained biological activity Good economy
4Strategy/AC/1998/JB/Å. Danielsson
Yields from Multistep Protein PurificationsYields from Multistep Protein Purifications
Number of steps
Yield (%)
95% / step
90% / step
85% / step80% / step75% / step
0
20
40
60
80
100
1 2 3 4 5 6 7 8
5Strategy/AC/1998/JB/Å. Danielsson
Input for Purification Protocol DevelopmentInput for Purification Protocol Development
Three phase strategy
Purificationprotocol
Required purityand quantity
Physical-chemical properties of target and
main contaminants
Source materialinformation
Separation techniqueknowledge
Scouting runs and optimization
Economy andresources
6Strategy/AC/1998/JB/Å. Danielsson
Protein PurificationProtein Purification
Analytical toolsA rapid and reliable assay for the target proteinPurity determination
(e.g. SDS-PAGE)Total protein determination
(e.g. colorimetric method)
7Strategy/AC/1998/JB/Å. Danielsson
Three Phase StrategyThree Phase Strategy
Purity
Step
Capture
Intermediatepurification
Polishing
Isolate product,concentrate, stabilize
Remove bulkimpurities
Achieve final purity.Remove trace impurities, structural variants,aggregates etc.
8Strategy/AC/1998/JB/Å. Danielsson
CaptureCapture
Resolution
Speed
Recovery
Capacity
Initial purification of the target molecule from crude or clarified source material
Concentration and stabilization (e.g. removal of proteases)
9Strategy/AC/1998/JB/Å. Danielsson
Intermediate PurificationIntermediate Purification
Resolution
Speed
Recovery
Capacity
Removal of bulk impurities
10Strategy/AC/1998/JB/Å. Danielsson
PolishingPolishing
Resolution
Speed
Recovery
Capacity
Final removal of trace contaminants, e.g. structural variants of the target protein
11Strategy/AC/1998/JB/Å. Danielsson
Three Phase Strategy - Ranking of Chromatography Techniques
Three Phase Strategy - Ranking of Chromatography Techniques
Technique Capture Intermediate Polishing
GF
IEX
HIC
AC
RPC
Considerations
Limited sample volumeLimited flow rate range
Protein ligand is sensitiveto harsh cleaning conditions
Use of organic solvents,loss of biological activity
12Strategy/AC/1998/JB/Å. Danielsson
Linking Chromatography Techniques into a Purification Protocol - General Rules
Linking Chromatography Techniques into a Purification Protocol - General Rules
Combine techniques with complementary selectivities (e.g. IEX, HIC and GF).
Minimize sample handling between purification steps (e.g. concentration, buffer exchange).
13Strategy/AC/1998/JB/Å. Danielsson
Linking Chromatography TechniquesLinking Chromatography Techniques
Technique End conditionsStart conditions
Small sample volume GF Diluted sampleBuffer change (if required)
Low ionic strength IEX High ionic strength orpH change
High ionic strength HIC Low ionic strength
Specific binding conditions AC Specific elution conditions
14Strategy/AC/1998/JB/Å. Danielsson
Linking Chromatography TechniquesLinking Chromatography Techniques
1. IEX HIC GF
2. AC GF
RPCIEX
3. HIC GFAC GF
4. (NH4)2SO4 HIC IEX GF
HIC GFIEX
5. GF GF (desalting) AC GF
15Strategy/AC/1998/JB/Å. Danielsson
Purity RequirementsPurity Requirements
Contaminants which degrade or inactivate the target protein (e.g. proteases), need to be reduced to “non-detectable” levels.
Contaminants which interfere with subsequent analyses need to be reduced to “non-detectable” levels.
It is better to “over-purify” than to “under-purify”.
16Strategy/AC/1998/JB/Å. Danielsson
• Therapy
• In vivo studies
• Crystallization for x-ray studies
• N-terminal sequencing of an unknown protein
• Most physical-chemical characterization methods
• Antigen for monoclonal antibody production
Extremely high High Moderate
Purity Requirements - Brief GuidelinesPurity Requirements - Brief Guidelines
17Strategy/AC/1998/JB/Å. Danielsson
Towards the Optimal Purification Protocol - Towards the Optimal Purification Protocol - Accounting for Target Protein Properties (1)Accounting for Target Protein Properties (1)Towards the Optimal Purification Protocol - Towards the Optimal Purification Protocol - Accounting for Target Protein Properties (1)Accounting for Target Protein Properties (1)
Target protein property Purification parameter affected
Stability window pH Ionic strength Co-factors Detergent concentration Organic solvents Other (light, oxygen etc.)
IEX conditions (also AC and RPC)
HIC conditions
selection of buffers, pH, salts, additives
buffer additives
RPC conditions
various
18Strategy/AC/1998/JB/Å. Danielsson
Towards the Optimal Purification Protocol - Towards the Optimal Purification Protocol - Accounting for Target Protein Properties (2)Accounting for Target Protein Properties (2)Towards the Optimal Purification Protocol - Towards the Optimal Purification Protocol - Accounting for Target Protein Properties (2)Accounting for Target Protein Properties (2)
Physical-chemical properties Charge properties (isoelectric point) Molecular weight Post-translational modifications Biospecific affinity
Target protein property Purification parameter affected
selection of IEX conditions
selection of GF medium
selection of group specific AC medium
selection of ligand for AC
19Strategy/AC/1998/JB/Å. Danielsson
Target Protein Stability WindowTarget Protein Stability Window
Determination of a suitable ammonium sulfate concentration and pH screening range for HIC
20Strategy/AC/1998/JB/Å. Danielsson
Target Protein PropertiesSelection of ion exchange conditionsTarget Protein PropertiesSelection of ion exchange conditions
0
5 6 7 8pH
mo
lecu
les
char
ge
+
-
Electrophoretic titration curve of chicken breast muscle using zymogram detection for creatine kinase
Target protein
Contaminants
21Strategy/AC/1998/JB/Å. Danielsson
G Protein Receptor Kinase PurificationG Protein Receptor Kinase Purification
Technique
Ppt
HIC
AIEX
CIEX
AC
Purificationfactor
Comment
7
20
2408
18647
• All buffers contain protease inhibitors
• All purifications done at +4o C
• Removal step, main contaminant is bound
• Elution buffer is used as starting buffer for next column
• 10 g homogenous proteinobtained
A. Tobin et al. (1996)J. Biol. Chem. 271, 3907-3916
Porcine cerebella homogenate
RESOURCE Q
Ammonium sulfateprecipitation
Butyl Sepharose Fast Flow
RESOURCE s
HiTrap Heparin
22Strategy/AC/1998/JB/Å. Danielsson
Rec -Mannosidase Purification from PichiaRec -Mannosidase Purification from Pichia
Technique Purificationfactor
Comment
• 83 g homogenous proteinobtained
Y.-F. Liao et al. (1996)J. Biol. Chem. 271, 28348-28358
• Capture with step gradient;730 mg of total protein applied
63
622
719
UF
GF
AIEX
HIC Phenyl Sepharose HP
Q Sepharose FF
Superdex 200 pg
Ultrafiltration
23Strategy/AC/1998/JB/Å. Danielsson
DNA Binding Protein PurificationDNA Binding Protein Purification
Technique
DNA-1 Sepharose
CIEX
AC
AC
AC
CIEX
Purificationfactor
Comment
5
8
4943
• General AC step for DNAbinding proteins
• Removal step, non-specificDNA binding activity removed
• Main purification step
• Final polishing, 20 g proteinobtained
J. Berthelsen et al. (1996) J. Biol. Chem. 271, 3822-3830
9
2447
• Rapid capture
HeLa cell nuclearextracts
SP Sepharose High Performance
Heparin Sepharose Fast Flow
DNA-2 Sepharose
Mono S
24Strategy/AC/1998/JB/Å. Danielsson
• Final polishing and purity check, 20 g obtained
Membrane Protein PurificationMembrane Protein Purification
Technique
AC
AIEX
CIEX
AC
CIEX
Purificationfactor
Comment
3
4
1442
• Negative step; contaminant removed
• Detergent exchange, volume reduction before AC
• Main purification step
T. White et al. (1995) J. Biol. Chem. 270, 24156-24165
6
242
• Step gradient, rapid concentratingcapture step
Placenta extract in1.5% Triton X-100
Blue Sepharose
DEAE Sephacel
SP Sepharose FF
Muc2 Sepharose
Mono S
25Strategy/AC/1998/JB/Å. Danielsson
Towards a General Protein Purification Protocol
Towards a General Protein Purification Protocol
A rapid method for obtaining milligram quantities of different recombinant proteins, for initial characterization studies
Semi-automated in ÄKTAexplorer, with pre-made method templates and BufferPrep
Ion exchange
STREAMLINE SP or DEAESP or Q Sepharose FF
Hydrophobic interaction
Phenyl Sepharose FF (high sub) Gel filtration
Superdex 75 prep grade
26Strategy/AC/1998/JB/Å. Danielsson
Towards a General Protein Purification Protocol - Results with E. coli r-ProteinsTowards a General Protein Purification
Protocol - Results with E. coli r-Proteins
Ion exchange
STREAMLINE SP or DEAESP or Q Sepharose FF
Hydrophobic interaction
Phenyl Sepharose FF (high sub) Gel filtration
Superdex 75 prep grade
Protein Expression Capture step(purified to homogeneity) Annexin V Extracellular STREAMLINE DEAE-Amylase Intracellular STREAMLINE DEAEanti-gp 120 Fab Periplasmic SP Sepharose Fast Flow
27Strategy/AC/1998/JB/Å. Danielsson
Shortcuts - Rapid Establishment of Milligram Scale Purification Protocols
Shortcuts - Rapid Establishment of Milligram Scale Purification Protocols
If a biospecific ligand is available: use AC as the main purification step.
If the purification is not intended to be scaled up: use high performance media (e.g. MonoBeads) throughout.
For “one-of-a-kind” purification of a protein e.g. for sequencing before gene isolation:sacrifice yield for purity by making narrow cuts.
If nothing is known about target protein and contaminants properties:try the IEX HIC GF combination.
Establish a fast and reliable assay for the target protein.
28Strategy/AC/1998/JB/Å. Danielsson
A Systematic Approach to Purification Development - Summary
A Systematic Approach to Purification Development - Summary
Develop assay methods Set the aims (purity and quantity) Characterize the target protein Use different separation principles Use few steps Limit sample handling between purification steps Start with high selectivity - increase efficiency Remove proteases quickly Reduce volume in early step Keep it simple!
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