HOW PROTEINS ARE PRODUCED & PURIFIED
- Select a source of protein
- Extract protein from the source (e.g. lysis)
- Separate proteins (preparative steps)
• Dialysis or ultrafiltration
• Differential solubilities
• Column chromatography
Size‐exclusion (gel filtration/permeation)
Ion‐exchange
Affinity (inc. purifying fusion proteins)
Hydrophobic interaction (HIC) - Assess purity and efficacy of purification (analytical steps)
• Protein quantitation to determine yield
• Analysis of separation to indicate purity
• Enzyme assay to indicate purity
Cells can be lysed/disrupted by
• Physical force (sonication,
pressure or grinding)
• Enzymes and/or osmotic pressure
• Detergents (e.g. SDS or tergitol)
How do you extract protein
1: Mix cells
2: Break open cells (lyse) to release contents of cell
(including protein)
3: Centrifuge cells
Supernatant containing protein is called “crude extract” (or Cell Free Extract, CFE)
What does the extraction buffers include
- Buffer (e.g. Tris: pH 7.5–8.5)
- Protease Inhibitors (e.g. EDTA: binds divalent metal ions required by metal proteases for activity)
- Reducing agents (e.g. ‐mercaptoethanol or DTT: inhibit disulphide bond formation)
- Salt
Reasons for separating proteins during purification:
1: To remove unwanted salts or ions: e.g. Dialysis & Buffer exchange using Ultrafiltration devices
2: To remove protein of interest from other contaminating proteins
What is dialysis
Procedure that separates proteins from small solutes by taking advantage of the protein’ larger size
Principle: passive diffusion through a semi‐ permeable membrane
How does dialysis purify
- Partially purified extract placed in semi-permeable membrane
- Suspended in large volume of buffered solution
- Membrane allows exchange of salt and buffer, not proteins
- Dialysis retains large proteins within the membranous bag
- Small molecules diffuse freely across membrane until they reach equilibrium with the solution outside the membrane
- Repeat until salt concentration is minimal
How does ULTRAFILTRATION of protein work
• Buffer exchange can be achieved by ultrafiltration
• Process involves: passing protein solution through a membrane filter under centrifugal force
• Molecules and salts smaller than MWCO pass through membrane
• Molecules larger than MWCO are retained within filter device
• Concentration of proteins can also be achieved by this
process
Ways to do Purification by Differential Solubilities
Precipitating proteins
Precipitating proteins using pH
- Proteins are least soluble at their isoelectric point (pI)
- Proteins have varying pI’s: can precipitate proteins differentially based on pI
- Can also use differences in pI in purification and analysis
- Precipitation of proteins using salt
• Protein solubility ‐ lowered at high [salt]
‘salting out’
• Salts can selectively precipitate proteins
(e.g. Ammonium sulfate (NH
4)2SO
4 ‐ highly soluble)
• Removed protein from those remaining in solution by precipitation and low speed centrifugation
• Can also be used to concentrate dilute proteins
What is the principle of Precipitation of proteins using salt
Principle • High [salt] removes hydrogen shell from
around protein
exposes ”hydrophobic” patches on protein surface
• Proteins aggregate via hydrophobic patches
• Water solvates proteins – forms hydration shell
• Polar/ionisable residues of protein interact with H2O through dipoles
• Most hydrophobic amino acid residues are found
within folded proteins, but some found on surface
• At hydrophobic patches, water forms ordered structure specific van der waals interactions with the protein
• Result: water is weakly bound at hydrophobic patches
• Addition of ammonium sulphate (NH4)2SO4 :
• dH2O drawn away from proteins
• co‐ordinated around the NH4+ and SO42‐ ions
• Exposed hydrophobic patches on protein interact form protein aggregates
• Protein precipitates
standard element used for COLUMN CHROMATOGRAPHY in separating protein
- Reservoir – supplies constant flow of buffer solution
- Stationary phase – porous solid matrix with chemical properties supported inside a column (glass or plastic)
- Mobile phase – buffered solution that flows through matrix of the stationary phase
- Eluent – solution that passes out of the bottom of the column
How do you do COLUMN CHROMATOGRAPHY
• Protein sample is layered on top of column
• Buffer is added from reservoir (constant flow)
• Proteins migrate through column, retarded to
different degrees depending on interaction with stationary phase
• Individual proteins separate from each other
• Eluted material is collected at bottom of column (in a specific elution volume, Ve)
How does Size-exclusion chromatography work
Separates proteins based on size
principle: movement within a liquid phase through a stationary porous medium
Column is matrix: beads made from cross‐linked polymers to form a porous matrix
Stationary phase: Solution inside the porous beads Mobile phase: Solution outside the bead • Large molecules cannot enter porous beads: have less volume to travel down the column • Small molecules enter beads: have access to a larger volume
Elution profile of proteins:
Vo = void volume: the elution volume of the molecules excluded from the size‐exclusion column
because they are larger than the largest pores
Ve = elution volume: measured from the chromatogram, relating to the molecular size of the eluted
molecule
Vt = total column volume: equivalent to the volume of the packed column bed
What can size exclusion chromatograhy use to determine
Mr
Advantage and disadvantage of size exclusion chromatograhy
Remove salt (or small mol wt) contaminants
e.g. Gel filtration on Sephadex G25, fractionation range (1 to 5 kDa)
Advantage: fast (<5 minutes)
Disadvantage: Only suitable for small volumes Dilute the protein sample
