Strategies in protein purification

1
Developing an efficient protein purification
scheme
2
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

3
Protein Purification - Aims

• Sufficient purity and quantity
• Maintained biological activity
• Good economy

4
Yields from Multistep Protein Purifications
Yield ()
95 / step
90 / step
85 / step
80 / step
75 / step
Number of steps
5
Input for Purification Protocol Development

Required purity and quantity
Three phase strategy
Purification protocol
Physical-chemical properties of target and main
contaminants
Separation technique knowledge
Source material information
Economy and resources
Scouting runs and optimization
6
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)

7
Three Phase Strategy
Achieve final purity. Remove trace impurities,
structural variants, aggregates etc.
Purity
Polishing
Remove bulk impurities
Intermediate purification
Isolate product, concentrate, stabilize
Capture
Step
8
Capture

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

Resolution
Speed
Capacity
Recovery
9
Intermediate Purification

• Removal of bulk impurities

Resolution
Speed
Capacity
Recovery
10
Polishing

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

Resolution
Speed
Capacity
Recovery
11
Three Phase Strategy - Ranking of Chromatography
Techniques
Considerations Limited sample volume Limited
flow rate range Protein ligand is
sensitive to harsh cleaning conditions Use of
organic solvents, loss of biological activity
Technique Capture Intermediate Polishing GF
IEX HIC AC RPC
12
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).

13
Linking Chromatography Techniques
Technique
End conditions
Start conditions
GF
Small sample volume
Diluted sample Buffer change (if required)
IEX
Low ionic strength
High ionic strength orpH change
HIC
High ionic strength
Low ionic strength
AC
Specific binding conditions
Specific elution conditions
14
Linking Chromatography Techniques
1. IEX HIC GF
2. AC GF
RPC IEX 3. HIC GF AC GF
4. (NH4)2SO4 HIC IEX GF
HIC GF IEX 5. GF GF
(desalting) AC GF
15
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.
• It is better to over-purify than to
  under-purify.

16
Purity Requirements - Brief Guidelines
Extremely high
High
Moderate

• Crystallization for x-ray studies
• N-terminal sequencing of an unknown protein
• Most physical-chemical characterization methods

• Antigen for monoclonal antibody production

• Therapy
• In vivo studies

17
Towards the Optimal Purification Protocol -
Accounting for Target Protein Properties (1)
Target protein property Purification
parameter affected
IEX conditions (also AC and RPC) HIC conditions
selection of buffers, pH, salts,
additives buffer additives RPC conditions various

• Stability window
• pH
• Ionic strength
• Co-factors
• Detergent concentration
• Organic solvents
• Other (light, oxygen etc.)

18
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

selection of IEX conditions selection of GF
medium selection of group specific AC
medium selection of ligand for AC
19
Target Protein Stability Window

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

20
Target Protein PropertiesSelection of ion
exchange conditions
Electrophoretic titration curve of chicken breast
muscle using zymogram detection for creatine
kinase
Contaminants
Target protein
21
G Protein Receptor Kinase Purification
Technique
Comment
Purificationfactor
Porcine cerebella homogenate
A. Tobin et al. (1996) J. Biol. Chem. 271,
3907-3916
Ppt
Ammonium sulfateprecipitation
7

• All buffers contain protease inhibitors
• All purifications done at 4o C

Butyl Sepharose Fast Flow
HIC
20
RESOURCE Q
AIEX

• Removal step, main contaminant is bound

2408
CIEX

• Elution buffer is used as starting buffer
  for next column

RESOURCE s
HiTrap Heparin
AC

• 10 mg homogenous protein obtained

18647
22
Rec a-Mannosidase Purification from Pichia
Technique
Comment
Purificationfactor
Y.-F. Liao et al. (1996) J. Biol. Chem. 271,
28348-28358

• Capture with step gradient730 mg of total
  protein applied

• 83 mg homogenous protein obtained

23
DNA Binding Protein Purification
Technique
Comment
Purificationfactor
HeLa cell nuclearextracts
J. Berthelsen et al. (1996) J. Biol. Chem. 271,
3822-3830
5
CIEX

• Rapid capture

SP Sepharose High Performance
AC
8

• General AC step for DNA binding proteins

Heparin Sepharose Fast Flow

• Removal step, non-specific DNA binding
  activity removed

AC
9
DNA-1 Sepharose
AC
2447

• Main purification step

DNA-2 Sepharose
CIEX
4943

• Final polishing, 20 mg protein obtained

Mono S
24
Membrane Protein Purification
Technique
Purificationfactor
Comment
T. White et al. (1995) J. Biol. Chem. 270,
24156-24165

• Step gradient, rapid concentrating capture step

AC
3

• Negative step contaminant removed

4
AIEX
CIEX
6

• Detergent exchange, volume reduction before AC

AC
242

• Main purification step

• Final polishing and purity check, 20 mg
  obtained

CIEX
1442
25
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 DEAE SP or Q
Sepharose FF
Hydrophobic interaction Phenyl Sepharose FF
(high sub)
Gel filtration Superdex 75 prep grade
26
Towards a General Protein Purification Protocol -
Results with E. coli r-Proteins
Ion exchange STREAMLINE SP or DEAE SP 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 a-Amylase Intracellular STREAMLINE
DEAE anti-gp 120 Fab Periplasmic SP Sepharose
Fast Flow
27
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
  isolationsacrifice yield for purity by making
  narrow cuts.
• If nothing is known about target protein and
  contaminants propertiestry the IEX HIC
  GF combination.
• Establish a fast and reliable assay for the
  target protein.

28
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!