Block A Lecture 2 - Methods for Protein Purification Flashcards
What is gel filtration?
It is a technique used to separate proteins based on size and shape. It can be used in protein purification.
The column contains porous beads which make up the stationary phase, which results in smaller proteins entering the pores of the bead, taking a longer path through the column, whereas larger molecules cannot enter the pores, and move faster through the column
(Slide 5)
What is the general buffer recommendation which is used in gel chromatography, and why is it used?
A buffer with intermediary ionic strength, which suppresses ionic effects and does not support hydrophobic interactions
(Slide 6)
What are 4 factors which can affect the resolution in gel filtration?
Diffusion (the particle size of the column material and flow rate)
The volume of the sample
Viscosity of the sample
Relative amounts of proteins to be separated
(Slide 7)
What are 3 uses of gel filtration?
Answers Include
Change of buffer (desalting)
Removal of reaction produce, co-factors or inhibitors
Removal of phenol red from culture medium prior to anion exchange chromatography
Fractionation
Determination of molecular mass
(Slide 9)
How is a calibration curve generated?
By injecting standard proteins of known molecule weights into the column which will partition between the pores of the Sephadex G-200 matrix
(Slide 11)
What is covalent chromatography?
Where the stationary phase is modified to contain reactive disulphides (such as 2-pyridyl disulphide) in order to enable covalent bonding to the target protein.
These bonds are stable and after the rest of the proteins have passed through the analytes can be eluted by changing the conditions (such as pH, temperature, or adding a ligand), which breaks the covalent bonds.
(Slide 13)
What has to be removed from the sample before covalent chromatography, and how is this done?
Low molecular weight thiols (such as glutathione), with can be removed by dialysis or gel filtration
(Slide 14)
How is binding monitored in covalent chromatography?
By measuring the absorption at 343 nm in the flow through
(Slide 14)
What are ion exchangers?
They contain functional groups capable of binding and releasing ions.
These sites are usually charged (positively or negatively) and can attract and hold ions of the opposite charge from the solution, separating them.
(Slide 18)
What is the difference between an anion and a cation exchanger?
An anion exchanger is a basic ion exchanger with positively charged functional groups whereas a cation exchanger is an acid ion exchanger which has negative functional groups
(Slide 18)
What is the difference between a strong and a weak ion exchanger?
A strong ion exchanger results in material being ionised over a broad pH-range. Capacity is not reduced by a loss of charged at high or low pH
Weak ion exchangers carry a weak base or acid as a functional group. They can lose charge at certain pH ranges.
E.g the carboxymethyl group of carboxymethyl ion exchanger is uncharged in pH values of under 5 and is therefore unavailable for exchange.
(Slide 18)
How can all proteins display cationic or anionic behaviour?
In acidic pH amino groups of lysine, arginine and histidine are protonated, so the protein can display cationic behaviour
In basic pH the negative charges of carboxyl groups of aspartate and glutamate take over, so the protein displays anionic behaviour
(Slide 20)
How does the isoelectric point tell us if a protein is basic or acidic?
If IP is above 8 - protein is basic.
If IP is below 6 - protein is acidic.
(Slide 20)
What are 3 requirements of a buffer used for ion exchanged chromatography?
Its ionic strength must be high enough to guarantee protein stability
It must have a high buffer capacity (pKa of buffer is not more than 0.5 pH units from the pH of the sample.)
Buffer ions should have the same sign as the charged groups of the ion exchanger, otherwise they would act as “counter-ions”
(Slide 22)
If freeze drying is planned in ion exchanged chromatography, what property should the buffer used have?
It should be volatile
(Slide 22)
How does a salt concentration gradient work in ion exchange chromatography?
The elution buffer initially has a low concentration of salt. This allows for weaker or less-affinity interactions between the target analytes and the ion exchange resin, meaning the analytes stay bound to the resin.
As the salt concentration is gradually increased, ions from the buffer (such as Na⁺ or Cl⁻) begin to compete with the analytes for the binding sites on the resin.
This results in analytes being displaced from the resin, with the higher the concentration of salt, the more analytes which are displaced, creating a concentration gradient
(Slides 23 - 25)
How does a pH gradient work in ion exchange chromatography?
pH is altered towards the isoelectric point of the substance to be eluted, as this is where many proteins have their lowest solubility.
Substances with weak interactions come off first.
This method results in aggregation via reduced repulsion - allowing proteins to be sorted
(Slide 26)
What is hydrophobic interaction chromatography?
A form of chromatography which separates proteins based on their hydrophobicity. It relies on the reversible interaction between the hydrophobic regions of a protein (or molecule) and hydrophobic groups immobilized on the stationary phase (the resin) within the column.
Protein binding is supported by an increased concentration of anti-chaotropic (neutral) salts, as they bind strongly with the water molecules, leaving less water available to “shield off” the hydrophobic ligands . Elution then occurs through a continuous reduction in salt concentration in the adsorption buffer.
(Slides 30 and 32)
What protein purification step is hydrophobic interaction chromatography well suited to follow?
Protein precipitation (via ammonium sulphate)
(Slide 31)
Why is hydrophobic interaction chromatography (HIC) suited to be the first chromatographic step?
As most biological starting material already has a high ionic strength (high conductivity)
(Slide 31)
Why is hydrophobic interaction chromatography suited to be used after ion exchange chromatography?
As the sample is already in a buffer with high ionic strength
(Slide 31)
What does increasing pH and lowering temperature do to the strength of hydrophobic interactions?
Increasing pH increases the strength of hydrophobic interactions whereas lowering the temperature reduce strength
(Slide 31)
How can high salt concentrations have a negative influence on selectivity in hydrophobic interaction chromatography?
As they can result in the precipitation of proteins on the column
(Slide 31)
What are 3 examples of additives, which in low concentrations, can reduce hydrophobic interactions?
H2O miscible (able to form a solution without separating) alcohols
Detergents
Chaotropic salts
(Slide 31)
What are 2 examples of salts which are commonly used in hydrophobic interaction chromatography?
Answers include:
Na2SO4
NaCl
(NH4)2SO4
(Slide 33)
In what situation does hydrophobic interaction chromatography yield a fairly concentrated protein?
In a low ionic strength solution
(Slide 34)
What is affinity chromatography?
A specific type of chromatography where molecules are separated based on their specific interactions with a biomimetic ligand or a biologically functional partner.
The ligand is immobilized onto a solid support (the stationary phase), and the target molecule is selectively captured through this interaction.
(Slide 36)
How is the adsorption mix generated in affinity chromatography?
Activated gel materials are usually used which allows covalent coupling of the ligand
(Slide 36)
What are 4 features a ligand has in affinity chromatography?
It has a reversible complex formation with the protein to be isolated
High specificity
Suitable dissociation constant to allow reversible complex formation without harming the protein (KD = 1 - 10 µM)
Chemical features which allow immobilisation to a matrix (stable in solvent used for coupling, at least 1 functional group for immobilisation, functional group must also not take part in the interaction involved in purification)
(Slide 37)
What are the classifications of ligand which can be used in affinity chromatography?
They are somewhere between monospecific and group specific and between low molecular to macromolecular size
(Slide 38)
What are 3 ways in which elution can occur in affinity chromatography?
By changing pH
Changing ionic strength
Changing structure or polarity (can be done by adding chaotropic salts, urea or guanidinium hydrochloride, polarity can be decreased by adding ethylene glycol or dioxane, or addition to detergents close to critical micelle concentration)
Specific elution by addition of a free ligand
(Slide 39)
What is immobilised metal ion affinity chromatography?
A type of chromatography that uses metal ions immobilized on a solid support to selectively bind proteins or other biomolecules that have a high affinity for these metal ions. It sorts proteins via metal binding groups.
This technique is especially useful for purifying proteins that contain specific metal-binding motifs or that have been engineered to include a metal-binding tag
(Slide 42)
What results in a higher yield in immobilised metal ion affinity chromatography (IMAC)?
The metal-chelator group binding stronger to the ion, the stronger the binding, the higher the yield
(Slide 42)
What are free co-ordination sites?
They refer to the vacant positions or spaces on a metal ion where a ligand can bind
(Slide 43)
Why do free coordination sites need to be purified before immobilised metal ion affinity chromatography?
To ensure efficient and specific binding of target molecules to the column
(Slide 43)
Why does a low pH result in elution in immobilised metal ion affinity chromatography?
As it results in changes in the charges of the functional groups and metal ions, affecting their interaction and possibly breaking it
(Slide 43)
Other than by lowering the pH, what is 1 other way in which elution can occur in immobilised metal ion affinity chromatography?
By addition of competitive ligands such as histidine or imidazole
(Slide 43)
