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
Protein Purification - Aims • Sufficient purity and quantity • Maintained biological activity • Good economy
100 80 60 40 20 0 1 2 3 4 5 6 7 8 Yields from Multistep Protein Purifications Yield (%) 95% / step 90% / step 85% / step 80% / step 75% / step Number of steps
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
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)
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
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
Intermediate Purification • Removal of bulk impurities Resolution Speed Capacity Recovery
Polishing • Final removal of trace contaminants, e.g. structural variants of the target protein Resolution Speed Capacity Recovery
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
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 Techniques Start conditions Technique End 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
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
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”.
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
Towards the Optimal Purification Protocol - Accounting for Target Protein Properties (1) Target protein propertyPurification 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.)
Towards the Optimal Purification Protocol - Accounting for Target Protein Properties (2) • Physical-chemical properties • Charge properties (isoelectric point) • Molecular weight • Post-translational modifications • Biospecific affinity Target protein propertyPurification parameter affected selection of IEX conditions selection of GF medium selection of group specific AC medium selection of ligand for AC
Target Protein Stability Window Determination of a suitable ammonium sulfate concentration and pH screening range for HIC
+ molecules charge 0 - 5 6 7 8 pH Target Protein PropertiesSelection of ion exchange conditions Electrophoretic titration curve of chicken breast muscle using zymogram detection for creatine kinase Contaminants Target protein
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 RESOURCE s CIEX • Elution buffer is used as starting buffer for next column HiTrap Heparin • 10 mg homogenous protein obtained AC 18647
Ultrafiltration UF Q Sepharose FF AIEX 63 HIC Phenyl Sepharose HP 622 719 Superdex 200 pg GF Rec a-Mannosidase Purification from Pichia Technique Comment Purificationfactor Y.-F. Liao et al. (1996) J. Biol. Chem. 271, 28348-28358 • Capture with step gradient;730 mg of total protein applied • 83 mg homogenous protein obtained
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 9 AC DNA-1 Sepharose 2447 AC • Main purification step DNA-2 Sepharose CIEX 4943 • Final polishing, 20 mg protein obtained Mono S
Placenta extract in1.5% Triton X-100 Blue Sepharose DEAE Sephacel SP Sepharose FF Muc2 Sepharose Mono S 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 6 CIEX • Detergent exchange, volume reduction before AC AC 242 • Main purification step • Final polishing and purity check, 20 mg obtained CIEX 1442
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
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
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 • 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!