Week 2 - Protein Chromatography Flashcards
WHAT IS CHROMATOGRAPHY?
A technique for analyzing or separating mixtures of gases, liquids or dissolved substances
What are the two stages of chromatography?
→ Stationary phase (matrix)
→Mobile phase
CHROMATOGRAPHY–USES
Forensics
- blood, arson, post mortem
Food regulation/testing
–horsemeat scandal, nutritional information
Athlete testing
–including horses! (combined liquid chromatography and MS)
Quality control
–alcohol, analysis of when a food spoils, water samples, contaminents
Pharmaceutical industry
–purification of antibodies, creating vaccines, purity of preparations
PRINCIPLES OF CHROMATOGRAPHY–MOBILE AND STATIONARY PHASES
- Mobile phase passed over stationary phase
- Sample–mobile phase
- Relative affinity for stationary phase allows for differentiation of sample
Two common methods of chromatography
column chromatography, planar chromotography
PRINCIPLES OF CHROMATOGRAPHY–TYPES OF CHROMATOGRAPHY
- Gas Chromatography (GC)
- Liquid Chromatography (LC)
- High-Performance Liquid Chromatography (HPLC)
- Thin-Layer Chromatography (TLC)
- Other chromatography techniques (exchange chromatography, affinity chromatography)
COLUMN CHROMATOGRAPHY - USES
- Separation of mixtures
- PurificationIsolation of active components
- Estimation of drugs in a formulation
- Isolation of active constituents
- Separation of diastereomers
COLUMN CHROMATOGRAPHY - VARIABLES
Dimension of the column: column efficiency can be improved by increasing length/width ratio of the column.
Particle size of column packing: think sand vs gravel
- Activity of the adsorbent
Temperature of the column
- speed of the elution increases at higher
Packing of the column
Quality of solvents
- solvents having low viscosities give better results
How does quality of solvents affect chromatography?
solvents having low viscosities give better results
How does dimension of the column affect chromatography?
column efficiency can be improved by increasing length/width ratio of the column.
PROTEIN EXTRACTION
Source of protein
–tissue or microbial cells We need to break open the cells–crude extract
Extraction of proteins
- Homogenization, Sonication, Freeze-thaw cycles, Organic solvents
PROTEIN PURIFICATION–PRELIMINARY STEPS
Precipitation and differential solubilization:
- Salting out (Ammonium sulphate)
- Detergents (Triton X-100, CHAPS)–dissolve cell membrane and keep protein in solution
Ultracentrifugation: Sub-cellular organelles
PROTEIN PURIFICATION
Need a pure preparation of a protein to determine properties or activity
PROBLEMOF PROTEIN PURIFICATION
Cells contain many proteins
- centrifugation to isolate organelles or fractions
- Proteins vary in size, charge, binding properties
Genetic engineering–modify proteins but alsofunction
PROTEIN CHROMATOGRAPHY
Crude extract fractionation purification of protein of interest..
Fractionation– pH, solubility, temperature …
- Salting out–depends on lowering solubility with salts e.g. ammonium sulphate
- Precipitates–centrifugation
PROTEIN CHROMATOGRAPHY - Dialysis
remove proteins from small solutes
PROTEIN CHROMATOGRAPHY - Column Chromatography - what does it use to seperate?
uses charge, size, binding affinity
PROTEIN CHROMATOGRAPHY - Differences from HPLC/GC
- Low pressure
- Low temperature
- Low flow rate
- Bigger columns/volumes
PROTEIN PURIFICATION - CHROMATOGRAPHY EXAMPLES
- Size-exclusion chromatography
- Ion-exchange chromatography
- Affinity chromatography
- Hydrophobic interaction chromatography
- Chromatofocussing
What is affinity chromatography based on?
Based on binding affinity
How are the beads in the column attached?
covalently attached ligand
PROTEIN CHROMATOGRAPHY & GENETIC ENGINEERING
- Many proteins do not bind a ligand that can be immobilized on a column
- BUT gene for almost any protein can be altered to express a fusion protein that can be purified by affinity chromatography
- Gene encoding the target protein is fused to a gene encoding a peptide or protein that binds a simple, stable ligand with high affinity and specificity–the tag.
- Tag sequences can be at amino or carboxyl terminus
PROTEIN CHROMATOGRAPHY & GENETIC ENGINEERING - EXAMPLES
GST tag
GST tag
GST enzyme binds to glutathione
Glutathione–immobilised on beads of agarose
- Retrieve target–wash with high concentration of salt or free glutathione–compete with immobilized ligand for GST binding
- Possible to remove the tag by protease cleavage
PROTEIN PURIFICATION - METHOD TO CHECK THE PRODUCT PURITY
find out the isoelectric point (pI) Approx mw
PROTEIN PURIFICATION - HOW TO CHECK THE PRODUCT PURITY
Electrophoresis
–Polyacrylamide gel
– a molecular “sieve”
–slowing migration of proteins in proportion to charge to mass ratio as below
SDS–PAGE
- Common method for estimating purity and mw
- Uses SDS
- Binding ratio 1.4x–nearly 1 molecule of SDS to each aa residue
- Sulfate moiety of bound SDS contributes to net negative charge–intrinsic charge of protein not significant
- SDS binding partially unfolds proteins–rodlike shapes
- Separation mainly on basis of mw
- Visualisation–Coomassie blue binds to proteins
CHROMATOFOCUSSING - OVERVIEW
- Chromatofocusing medium equilibrated with a start buffer at a pH slightly above the highest pH required.
- Sample is applied to the chromatographic column with the start buffer
- Elution buffer is passed through the column and begins to titrate the amines on the medium and the proteins–gradient pH develops
- Proteins in the sample that are at a pH above their pI are negatively charged and retained near the top of the column
- The ones having their pH below pI begin to migrate down and bind to that part of the column where the pH is above their pI
CHROMATOFOCUSSING
- Binding dependent on the surface charge of the protein
- Uses ion exchange resins
- Uses FPLC
- Elutes bound species by altering the pH of the buffer
- Proteins elute in order of their isoelectric points.
2D GEL ELECTROPHORESIS - So why not combine SDS page and isoelectric focussing?
- More sensitive than either alone..
- separates proteins of identical molecular weight that differ in pI, orproteins with similar pI values but different mw
POST CHROMATOGRAPHY PROCESSING
CONCENTRATION
- Lyophilization aka. freeze drying
- Ultrafiltration
- Chromatographic concentration
- Precipitation
POST CHROMATOGRAPHY PROCESSING
YIELD AND ANALYSIS TECHENQUES
- Enzyme assay
- Protein assay
- SDS-PAGE
- Western Blotting
POST CHROMATOGRAPHY PROCESSING
YIELD AND ANALYSIS
Simulation
SDS-PAGE & immunoblotting
POST CHROMATOGRAPHY PROCESSING
RECOMBINANT PROTEINS
- Manipulated form of native protein
- Generated in various ways in order to increase production of proteins, modify
- Coding sequence for the protein of interest is isolated and cloned into an expression plasmid vector.
- Most recombinant proteins for therapeutic use are from humans but are expressed in microorganisms such as bacteria, yeast, or animal cells in culture.
- intron-free version of the gene is often made by converting the mRNA into cDNA
- But cDNA lacks regulatory regions so expression vectors provide promoter, ribosome-binding site, and terminator sequences
WHAT ARE RECOMBINANT PROTEINS USED FOR?
Lab techniques–
- ELISA–matched antibody pairs–standards
- Western Blot–positive controls
- Immunohistochemistry
- Enzyme assays
- Cellular responses to stress and disease
- In animal models–can help with identifying therapeutic targets
EXAMPLES OF RECOMBINANT PROTEINS
First use–insulin (1982)
Now–recombinant hormones, interferons, interleukins, growth factors, tumor necrosis factors, blood clotting factors, thrombolytic drugs,treating major diseases
Enzymes–animal feed enhancement
Lactic acid bacteria–used for fermenting foods, now adapted for use in human/animal digestion and nutrition
ADVANTAGES OF RECOMBINANT PROTEINS
Ethical considerations
Quick
Cost
Scaling
DISADVANTAGES OF RECOMBINANT PROTEINS
Contamination, e.g. proteasesInactive protein, e.g. inclusion bodies
Small proteins only
Lack of post-translational modifications
What occurs at the stationary phase?
catch different parts of solution or gas we are measuring eg. paper where we pass liquid over it
What occurs at the column?
load sample with solution
let it run through
What is column packing?
the thing that is absorbing packing material
How do you equilibrate a column?
we need to equilibrate column
this is done by putting solvent through column eg. water or petrol
so column is bathed in whatever the solute is going to dissolve in
What is silica good for?
polar compounds
What effects does dialysis use?
uses osmotic effects to draw out small solutes
What solid phase is used in ion exchange chromatography?
resin - charged
How do proteins move in ion exchange chromatography?
proteins move through depending on charge
What happens if we have a negative charge on our resin (cation exchange) - ion exchange chromatography
proteins with negative charge move faster ad come out earlier
How does size-exclusion chromatography separate proteins?
separates protein molecules by size
- larger molecules come out earlier as smaller molecules are trapped in the beads
How does affinity chromatography bind to molecules?
by forming ligands
- they trap molecules of interest as it goes down the column
What does gradient elution involve?
change binding conditions
proteins separated out differently
2 peaks formed
Matrix - Affinity Chromatography
for ligand attachment
matrix should be chemically and physically inert
Spacer Arm - Affinity Chromatography
used to improve binding between ligand and target molecules by overcoming any effects of steric hindrance
Ligand - Affinity Chromatography
molecule that binds reversibly to a specific target molecule or group of target molecule
Affinity Chromatography - Step 1
affinity medium is equilibrated in binding buffer
Affinity Chromatography - Step 2
sample is applied under conditions that favour specific binding of the target molecules(s) to a complementary binding substrate (the ligand)
target substance bind specifically but reversibly to the ligand and unbound material washes through the column
Affinity Chromatography - Step 3
target protein is recovered by changing conditions to favour elution of bound molecules
elution is performed specifically using a competitive ligand or non-specifically by changing the pH, ionic strength or polarity
target protein is collected and purified, concentrated form
Affinity Chromatography - Step 4
affinity medium is re-equilbrated binding buffer
Process of affinity chromatography
equilibrium
adsorption of sample and elution of unbound material
wash away unbound material
elute bound protein(s)
re-equilibration
What happens in Hydrophobic Interaction Chromatography (HIC)
salt concentration is lowered gradually and samples will elute from column in order of hydrophobicity
What is on the y axis of a chromatography analysis graph?
absorbance
What is on the x axis of a chromatography analysis graph?
column volumes (CV)
What is added to a Hydrophobic Interaction Chromatography (HIC) and why?
as we move along, more tightly bound molecules
therefore we change salts
Why do we add Ammonium Sulphate to Hydrophobic Interaction Chromatography (HIC)?
helps precipitate out any addition of proteins
What does increasing salt concentration on Hydrophobic Interaction Chromatography (HIC) allow?
better binding
Tag
bind gene encoding on target protein so we recognise it at the end
tag can be put at either ends
Why do we use tags?
we use its properties to separate our target
Polyarylamide gel
acts as molecular signal to filter different proteins
SDD
type of detergent
Page
type of polyacrylamide gel
What direction foes SDS-PAGE run?
run vertically rather than horizontally
Kamasi Blue
very good for binding to protein
tells us where proteins are coming out of gel
What can we use to look at where protein is sitting and what the isoelectric point is?
staining
What is the Western blotting?
bind proteins of interest with primary antibodies and a conjugate with a dye
What does the plasmid vector do in recombinate protein chromatography?
take sequence and amplified part of reproductive processes
Where do you get recombonant proteins from?
from humans
What do we need if we put a sequence of interest into vector?
we need promotor regions
What’s a disadvantage of sequence of interest into vector?
cDNA does not have regulatory genes
How do beads in the column attach in affinity chromatography?
Beads in the column - covalently attached ligand
Process of affinity chromatography
Any protein with affinity for the ligand binds to the beads - migration retarded
Proteins that do not bind flow more rapidly through the column
Bound proteins eluted by a solution containing either a high concentration of
Free ligand competes with the ligand attached to the beads, releasing the protein product that elutes from the column is often bound to the ligand used
