Developing an efficient protein purification scheme

1Strategy/AC/1998/JB/Å. Danielsson

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

Developing an efficient protein purification schemeDeveloping an efficient protein purification scheme


Protein Purification - AimsProtein Purification - Aims

Sufficient purity and quantity Maintained biological activity Good economy


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


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


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)


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.


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)


Intermediate PurificationIntermediate Purification

Resolution

Speed

Recovery

Capacity

Removal of bulk impurities


PolishingPolishing

Resolution

Speed

Recovery

Capacity

Final removal of trace contaminants, e.g. structural variants of the target protein


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


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).


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


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


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”.


• 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


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


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


Target Protein Stability WindowTarget Protein Stability Window

Determination of a suitable ammonium sulfate concentration and pH screening range for HIC


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


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


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


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


• 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


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


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


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.


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!

Developing an efficient protein purification scheme

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