Target Protein Properties
Selection of ion exchange conditions Electrophoretic titration curve of chicken breast muscle using zymogram detection for creatine kinase
Developing an efficient protein purification scheme
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Developing an efficient protein purification scheme
Introduction
IEX HIC AC Protein ligand is sensitive to harsh cleaning conditions Use of organic solvents, loss of biological activity
RPC
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Target Protein Stability Window
Determination of a suitable ammonium sulfate concentration and pH screening range for HIC
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It is better to “over-purify” than to “under-purify”.
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Purity Requirements - Brief Guidelines
Extremely high
• Therapy
IEX conditions (also AC and RPC) HIC conditions selection of buffers, pH, salts, additives buffer additives RPC conditions various
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Protein Purification
Analytical

tools
A rapid and reliable assay for the target protein Purity determination (e.g. SDS-PAGE) Total protein determination (e.g. colorimetric method)
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Input for Purification Protocol Development
Required purity and quantity
Three phase strategy
Purification protocol
Separation technique knowledge
Recovery
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Three Phase Strategy - Ranking of Chromatography Techniques
Technique GF Capture Intermediate Polishing Considerations Limited sample volume Limited flow rate range
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Protein Purification - Aims
Sufficient
purity and quantity
Maintained Good
biological activity
economy
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Linking Chromatography Techniques into a Purification Protocol - General Rules
Combine
techniques with complementary selectivities (e.g. IEX, HIC and GF). sample handling between purification steps (e.g. concentration, buffer exchange).
+ molecules charge
Contaminants 0 Target protein 5 6 7 pH 8
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G Protein Receptor Kinase Purification
• In vivo studies
High
• Crystallization for x-ray studies
• N-terminal sequencing of an unknown protein • Most physical-chemical characterization methods
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Three Phase Strategy
Purity
Achieve final purity. Remove trace impurities, structural variants, aggregates etc. Remove bulk impurities
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Polishing

Final removal of trace contaminants, e.g. structural variants of the target protein
Resolution
Speed
Capacity
selection of IEX conditions selection of GF medium selection of group specific AC medium selection of ligand for AC
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Target protein property

Purification parameter affected
Stability window pH Ionic strength Co-factors Detergent concentration Organic solvents Other (light, oxygen etc.)
Low ionic strength
IEX
High ionic strength or pH change
Low ionic strength
High ionic strength
HIC
Specific binding conditions
AC
Specific elution conditions
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Yields from Multistep Protein Purifications
Yield (%)
100 80 60 40 20 0 1 2 3 4 5 6 7 8
95% / step 90% / step
85% / step 80% / step 75% / step
Number of steps
Towards the Optimal Purification Protocol Accounting for Target Protein Properties (2)
Target protein property

Purification parameter affected
Physical-chemical properties Charge properties (isoelectric point) Molecular weight Post-translational modifications Biospecific affinity
GF
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Purity 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.
Polishing
Isolate product, concentrate, stabilize
Intermediate purification
Capture
Step
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Capture

Initial purification of the target molecule from crude or clarified source material Concentration and stabilization (e.g. removal of proteases)