Lecture 8 Flashcards
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
