Lecture 3: Chromatography Flashcards
What is chromatography? What do we need to consider when we use it?
Chromatography relies on proteins differentially interacting with chemical groups attached to beads and resins. There are many things we need to consider:
• Size.
• Does the protein binds to a known ligand?
• Can the protein be equipped with tags?
• pI.
• How selective is the column? Will it bind to contaminants?
• What is the capacity of the column? We want to bind as much protein as we can and not leave free surface area which can bind to contaminants.
• What is the resolution?
• How compatible is this step with the next one?
Chromatography columns can be run in a gravity flow or in a packed column with controlled flow. We normally start a purification process with affinity chromatography, which can use ligands, tags or antibodies.
How does the histidine tag work?
The his-tag contains 6 his residues or more.
• It interacts with a resin containing metals in immobilised metal affinity chromatography (IMAC).
• The most commonly used resins are Ni2+ or Co2+. They are chelated by an NTA group attached to a resin.
• Each metal has 2 free coordination sites free to interact with 2 His residues.
• After washing to get rid of contaminants, the protein is eluted with imidazole. This molecule can compete with the tag to bind to the metal.
• Its advantages are that it is small and simple, and it rarely interferes with protein function. It doesn’t need to be removed. It also works in denaturing conditions (urea or guanidine).
• On the other hand, it is not a very specific interaction and proteins with surface His clusters can non-specifically interact.
How does the GST tag work?
Glutathione-S-transferase is a larger tag.
• The tag specifically interacts with glutathione.
• A glutathione resin can be used for purification.
• Free glutathione (pH adjusted to 8) is used to elute the protein.
• Its advantages are that it is very specific (fewer contaminants), elution conditions are mild, it is quick and easy, and reagents are all commercially available.
• However, it is large, and it must be removed. It is also a big extra protein for a cell to make. Furthermore, GST dimerises which will lead to false results if one is looking at the multimeric state of fusion proteins.
How does the MBP tag work?
Maltose binding protein is another large tag which can be used.
• MBP binds specifically to amylose columns.
• Maltose can also be used to elute the protein.
• MBP can increase the solubility of fusion proteins.
• It is quick, easy and cheap. It has mild elution conditions and it binds very efficiently. The solubility change is also an added advantage.
• However, again it is a large extra protein to be added. It needs to be removed for many applications.
How can ligand resins be used?
It is not always possible or desirable to tag a protein. In this case, we can use ligand resins as an alternative.
• Sometimes we need an authentic protein from native source, with no tags or changes.
• If the protein has a specific ligand, then we can exploit this by making a resin with the said ligand.
• For example, the β-adrenergic receptor can be purified with alprenolol. Alprenolol is an antagonist of the receptor with nanomolar affinity.
What are immunoaffinity resins?
Immunoaffinity methods use antibodies to bind to the protein of interest.
• The antibody can be created by injecting the protein into a mouse or rabbit.
• The resins are very specific.
• Elution is difficult. We accomplish it by using a competitive peptide. Sometimes pH changes are required as well.
• Resins are made by chemical linkage.
• Protein A and protein G can be used to bind to the immunoglobulin. The proteins are bound to an agarose bead.
• Sepharose and cyanogen bromide can be used to attach proteins to agarose resin.
What are the advantages and disadvantages of tailored resins?
Tailored resins refer to ligand resins and immunoaffinity resins.
Advantages
• They are highly specific.
• They don’t require tagging.
• The protein can be extracted from natural sources, with all the standard modifications and processing.
• The proteins may elute in complex with binding partners. The binding partners can then be identified.
Disadvantages
• Difficult to elute.
• Tailored approach required every time.
• Time consuming and expensive.
What is ion exchange chromatography?
IEC is a method which separates proteins based on their surface changes.
• Charged resins make electrostatic interactions with the protein of interest.
• These interactions can be broken using salts like NaCl. The Na and Cl ions act as counter ions. They block interactions and lead to elution.
• Different proteins will bind with different strengths and therefore elute at different NaCL concentrations.
• Resins can be anion exchanges (with a strong positive charge) or cation exchanges (with a strong negative charge).
• The resins can be weak or strong. Strong resins are ionised over all working pH values. Weak resins are ionised over a specific range of pH values.
• Q sepharose is a strong anion exchanger.
• Sp sepharose is a strong cation exchanger.
How is ion exchange chromatography performed?
- It is important to know the pI of a protein.
- If the pH > pI, the protein will have a net negative charge and bind to an anionic resin.
- If the pH < pI, the protein will be positively charged and bind to a cationic resin.
- We must remember that proteins have many ionisable groups on their surfaces, the will have charge patches of different charges.
- We generally work one pH unit away from the pI.
- We can predict pI from AA sequence. However, the environment will influence side chains, so this is only an estimate.
- We chose the appropriate resin and buffer combination.
- The protein is put in the binding buffer which is at the correct pH and has little NaCl.
- The protein is allowed to interact with the resin and its washed with a low salt buffer.
- The protein is eluted with a pH change of a gradient of NaCl.
What is chromatofocusing?
Chromatofocusing is a very powerful method which can separate proteins by their pI.
• It is very rarely used.
• It relies on a pH gradient being created on the column. A weak ion exchange resin interacts with a strong buffer.
• Proteins will move down the pH gradient, binding and unbinding to the resin. Proteins move at different rates depending on pI.
• It has good separation but low capacity.
What are the pros and cons of ion exchange chromatography and chromatofocusing?
Advantages
• No modification of the sample.
• Versatile, can be used for anything.
• High capacity.
• Chromatofocusing can separate very similar molecules which differ just in surface charges (e.g. phosphorylation variants).
Disadvantages
• Relatively low selectivity for the average protein because many proteins have similar pI values.
What is hydrophobic interaction chromatography?
HIC is a method which can be used to separate proteins based on the amount of hydrophobicity in their surfaces.
• Hydrophobic interactions are strongest in buffers with high ionic strength. This is because water molecules released by the interaction will interact with ions in a favourable manner.
• We can therefore bind the protein to the resin in high salt concentrations (around 1-3 M salt) and elute with a decreasing salt gradient.
• It is ideal for ammonium sulphate precipitated samples.
What is reverse phase chromatography?
RPC is another method that can separate molecules based on hydrophobicity.
• Proteins are loaded in an aqueous buffer. This is often water with a small amount of TFA (0.1%) to control pH.
• The proteins are eluted with an increasing concentration gradient of an organic solvent such as acetonitrile.
• This method might not be great for proteins as organic solvents often cause denaturation.
• This technique is often used for peptides. A C18 containing resin is used.
• The protein is eluted with a gradient of acetonitrile.
How can we separate proteins based on size?
Size exclusion (or gel filtration) chromatography can be used to separate proteins based on size and shape.
• A porous resin is used. Molecules smaller than the pores will enter into them and be slowed down.
• Molecules bigger than the pores will pass around the beads and move faster.
• We can use different columns with different size pores in the beads.
• The sample does not bind to the resin and is not concentrated. The volume loaded onto the column affects the degree of separation of peaks emerging (resolution).
• Big columns have more separation, but they also take longer to run.
• Large columns are used for purification and small columns are used for analytical work.
• The sample is first concentrated to a small volume (less than 5% of the column volume).
• The sample is loaded onto the column in a continuous flow of a buffer in which the protein is stable.
• SEC is very useful for removing aggregated protein.
• We use a series of standards for calibration.
• A SEC has a void volume (V0¬). It is the volume outside the beads. Very large macromolecules elute here.
• The column also has a total volume (VT), where very small things elute.
• The protein will elute between these two volumes if the correct column is being used.
• We create a calibration curve and use it to estimate the mass of a macromolecule based on its elution volume.
• It is simple to use, and various resins are available for different particles.
• The disadvantages are that the sample must be concentrated to get good separation. The elution volume is also dependent on the shape of the protein.
How do we create a purification strategy?
We must combine multiple chromatography methods in order to create a coherent strategy.
• We want as few steps as possible because each step loses sample. Proteins can also have limited stability, so we want to be fast.
• We usually start with an affinity method. They’re quick and massively increase purity. They can also remove proteases.
• We normally finish with gel filtration. This removes aggregated or misfolded proteins.
• We can tailor the middle steps in order to remove specific contaminants.
