Structural Biochemistry/Proteins/Purification/Affinity chromatography
Affinity chromatography is an applicable technique used to purify proteins. It is performed depending on the advantage of the high affinity of proteins for specific chemical groups. Affinity chromatography was discovered by Pedro Cuatrecasas and Meir Wilcheck in 1968.
This process is generally used to isolate interested protein from the pool of proteins. A column is filled with beads that contain covalently attached glucose residues. It is taken in consideration that these residues are chosen corresponding to the target protein. As the protein mixture is poured into the column, the proteins will travel down through the beads. The target protein will be recognized and get trapped to the column by covalent bond because of its affinity for glucose. The rest of proteins will run down to the column and be separated. The portion of buffer needed to be added to the column to wash out completely the unbounded protein. Lastly, a concentrated solution of glucose with be added to separate the target protein from the column-attached glucose residues.
The starting part included an undefined heterogeneous mixture of molecules in solution. The desired molecules will have defined property which can be exploited during the affinity purification process. The process is a setup in which the target molecule becoming trapped on stationary medium. The non-target heterogeneous mixture will not become trapped due to its unbounded ability. The solid medium can then be removed from the mixture, washed multiple times, and the target molecule released from the entrapment in a process known as elution with high concentration of specific chemicals or altering the conditions to decrease the binding ability. Also, it is important that the reaction is carried in an appropriate pH; otherwise, it may reduce the affinity and change the conformation of the proteins, preventing the target protein to bind to the residues as expected.
Affinity chromatography is a powerful means of isolating transcription factors, proteins that regulate gene expression by binding to specific DNA sequences. A protein mixture is percolated through a column containing specific DNA sequences attached to a matrix. Proteins with a high affinity for the sequence will bind and be retained. In this instance, the transcription factor is released by washing with a solution containing a high concentration of salt.
In general, affinity chromatography can be effectively used to isolate a protein that recognizes group X by:covalently attaching X or a derivative of it to a column, adding a mixture of proteins to this column, which is then washed with buffer to remove unbound proteins, eluting the desired protein by adding a high concentration of a soluble form of X or altering the conditions to decrease binding affinity. Affinity chromatography is most effective when the interaction of the protein and the molecule that is used as the bait is highly specific.
- 1 Utilization
- 2 Combinatorial Chemistry
- 3 Immunoaffinity Chromatography
- 4 Immobilized metal ion affinity chromatography (IMAC)
- 5 Interaction materials
- 6 First technique
- 7 Elution Methods
- 8 pH elution method
- 9 Ionic strength changing method
- 10 Competitive agents elution
- 11 Reduced polarity of eluent
- 12 Chaotropic eluents
- 13 Histidine tag
- 14 Glutathione S-transferase (GST) tags
- 15 Lectin Affinity Chromatography
- 16 Advantages and disadvantages
- 17 Reference
Affinity chromatography is mainly used in biochemistry to
• Purify certain proteins from a mixture
• Reduce the amount of a certain protein molecule in a mixture of multiple proteins
• Discover the affinity of substances to biological compounds, in this case protein.
Affinity chromatography can also be used in combinatorial chemistry (in-vitro evolution), in which you can imitate the process of evolution by creating large sets of molecules and selecting for a specific function. In this case, you start from a diverse population of molecules, then select for particular proteins, and reproduce that molecule. For instance, starting with a randomized pool of RNA segments and an ATP affinity column, you would apply the RNA pool to the top of the column. Next, you would allow the selection of ATP-binding molecules to occur, eluting from the RNA pool all the segments that did not bind to the ATP. Then to elute the bound RNA molecules, you apply ATP to the top of the column. This isolates the selected RNA molecules that are bound to ATP. You can expand this selection by using different salt concentrations, with increased salt concentrations being more selective.
An example of immunoaffinity chromatography is by the use of blood antibodies. Blood antibodies can be purified by use of affinity purification form the blood plasma (serum). If there is antibodies in the blood plasma that are against some particular antigen we can use this for the antigen purification by using affinity. A common example to see if an organism is immune against a GST-fusion protein by observing if it produces antibodies against GST tag and the fusion-protein. Foremost, the GST affinity matrix is allowed to bind to the blood plasma. Allowing the blood plasma to bind helps remove antibodies against the GST. Separation of the blood plasma form the solid helps it bind to the GST-fusion protein matrix which in turn traps the antigen that is recognized by the antibody in the solid support. Using low pH ( pH 3 ) buffers for elution helps obtain the desired antibodies. Collection of the eluate is mostly done in phosphate buffer to neutralized the low pH.
Immobilized metal ion affinity chromatography (IMAC)
IMAC is particular based on coordination with covalent bonds form amino acids to metals. The concept of this technique is to keep in the column proteins with affinity to the metal ions which get immobilized inside the column. Iron, gallium or zinc can be used to purify phosphorylated proteins or peptides. Common metals for binding histidine are copper, cobalt, and nickel. DNA recombinant technologies are use since many natural occurring proteins do not have affinity to metal ions.
These are typical biochemical interactions in nature that have been used extensively in affinity chromatography:
• Enzyme will bind to substrate analogue, inhibitor, and cofactor
• Lectin will bind to polysaccharide, glycoprotein, cell surface receptor, cell
• Antibody will bind to antigen, virus, cell
• Nucleic acid will attach to complementary base sequence, histones, nucleic acid polymerase, and nucleic acid binding protein
• Hormone, vitamin will bind to receptor, carrier protein
• Glutathione will bind to glutathione-S-transferase or GST fusion proteins
• Metal ions will attach to Poly (His) fusion proteins, native proteins with histidine, cysteine and tryptophan residues on their surfaces.
Commonly, affinity chromatography will be done through column chromatography. First of all, the binding ability of protein must be studied. Then, the solid medium modified with the binding material is packed in a chromatography column. Then, the initial mixture that contained desired proteins was added through the column to allow binding to occur. A wash buffer was gradually added to the addition of mixture. The elution buffer subsequently removes unbounded protein from the column and collected.
There is no generally applicable elution methods for all affinity media. When substances are very tightly bounded to the affinity medium, it may be useful to stop the flow after applying eluent, usually 10 minutes to 2 hours is referred, before continuing the elution process. This extra time helps to improve recovery percentage of bounded protein.
Forces that maintain the complex of substrate and bound substances include electrostatic interactions, hydrophobic interactions, and hydrogen bonding. Agents that deteriorate these interactions may be expected to function as efficient eluting agents. The optimal flow rate to achieve efficiency may vary according to the specific interaction.
pH elution method
This is one of most common techniques that are used to remove bounded protein from the ligands. A change in pH alters the charged groups on the ligands and/or the bound protein. This change may directly affect the binding sites and reducing their affinity. On the other hand, a change in pH can cause indirect modification in affinity by altering in conformation of proteins. A sudden decrease in pH is one of the most common methods to elute bounded proteins. The chemical stability of the ligand and target proteins determines the limitation of pH change. The column should always return to neutral pH immediately after the elution to avoid irreversible denature of proteins.
Ionic strength changing method
Changing ionic strength of buffer solution will alter the specific interaction between the ligand and target protein. This method is a mild elution using a buffer with increased ionic strength usually sodium chloride, applied as a linear gradient or in steps.
Competitive agents elution
Selective eluents are often utilized to separate substances on a specific medium or in the presence of high binding affinity of the ligand/target protein interaction. The eluting agent competes either for binding to the target protein or for binding to the ligand. This is an example of competitive inhibitors that occur in nature. Substances may be eluted either by a concentration gradient of a single eluent. In this method, the concentration of competitive agents should be added equally to the concentration of the coupled ligand. However, if the free competing compound binds more weakly than the ligand to the target protein, use a higher concentration of competitive agent to achieve efficiency in elution.
For example of competitive affinity chromatography, There is R1a protein. The target R1a protein bind to cAMP resin. The interaction between R1a protein and cAMP would separate by using cGMP elution buffer. This cGMP compete with target protein, however the elution buffer which contains high concentration of cGMP would bind to resin more. The separated R1a protein will eluted out.
Reduced polarity of eluent
Conditions are used to lower the polarity of the eluent promote elution without inactivating the proteins. Dioxane or ethylene glycol are typical of this type of eluent.
In case of other elution methods fail, deforming buffer solution, which alters the structure of proteins, can be used to achieve separation of ligand and target proteins. Typical chaotropic agents are guanidine hydrochloride and urea. Although this method will yield the highest percentage of recovery, chaotropes method should be avoided whenever possible since they are to denature the eluted protein.
Affinity chromatography can be performed using a number of different protein tags. One of the common tag using in laboratory is poly-hisitidine. Its shortness in length prevents altering the conformation of the tagged protein. Histidine tagging is favorable because it is very specific, allowing for a high level of purification.
The gene which encodes for a specific protein is first modified to include the tag. A string of histidine residues may be added to the amino or carboxyl terminus of the expressed protein. The tagged proteins are then passed through a column of beads containing covalently attached, immobilized nickel(II) (Ni 2+.) This His-tag binds tightly to the immobilized metal ions because the side chain of Histidine, imidazole, has a specific binding affinity to metal ions (in this case, nickel II). As a result, the desired protein is binded tightly to the beads while other proteins flow through the column easily. Even other, non-desired proteins, that have Histidine side chains will flow through because they do not have as many as the desired, tagged protein, which would have about 6 adjacent Histidine residues. The protein can then be eluted from the column by addition of imidazole or some other chemicals that bind to the metal ions and displace the proteins. The presence of desired proteins can be verified through enzyme-linked immunosorbent assay (ELISA).
Nickel resin regeneration
In recombinant DNA, histidine tag on the desired protein and Nickel resin are commonly used to purify desired protein via affinity chromatography. That is, histidine has strong affinity towards the nickel resin which does not flow through the column. Undesired proteins do not have the designed histidine sequences hence could not bind to Nickel resin; those protein flow though the column. During elution, we add a relatively high concentration of imidazole buffer. Imidazole compete with our desired protein to bind with the nickel resin. In practice, Nickel resin is rather expensive. Regeneration of Nickel resin is essential. It involves several steps. First, there are possible left over protein remained on used Nickel resin; these left over protein are denatured and washed away using Guanidinium chloride and corresponding buffer. The Nickel resin is washed with Milli Q water and increasing concentration of Ethanol. One essential step in Nickel resin regeneration is recharging the Nickel. We first remove the Nickel with EDTA, which is a hexa-dendate compound that releases the Nickel ion. The Resin would then turn white without Nickel. Then the resin is recharged with high concentration of nickel salt to obtain our slightly green resin.
GST has an affinity for glutathione, which is available immobilized as glutathione agarose. An excess amount of gluthione is used to displace the tagged protein for elution. Together with the histidine tags, the purification of recombinant proteins like GST tags is the most common use of affinity chromatography.
GST are enzymes involving cellular defense against electrophillic compounds. It has hing affinity and specificity to bind with glutathinone. The strength and selectivity of this interaction allow GST tagged proteins to be purified by the glutathione-based protein resins. The glutathione resins selectively bind to GST-tagged proteins effectively, allowing the specific protein of interest be separated from the mixture at high efficiency.
GST is a 35-KDa protein, it has small peptides. It is this characteristic which allows one to perform GST-protein purification quickly without degradation by proteases and minimize sample loss.GST will lose its ability to bind Glutathione resin when it is denatured, therefore, strong denaturant such as Guanidine-HCl and urea cannot be added in the buffers.
Lectin Affinity Chromatography
Lectin protein, for example concanavalin A which is originally extracted from the jack-bean Canavalia ensiformis, binds specifically to some certain structures in sugars. Lectin affinity chromatography is one kind of affinity chromatography in which the plant protein concanavalin A is purified by passing a crude extract through a column of beads containing covalently attached glucose residues. Since it has affinity to glucose, concanavalin A will bind to this type of column. A concentrated solution of glucose is then added to remove the bound concanavalin A from the column.
Advantages and disadvantages
• Affinity chromatography is a fairly achievable technique because of the great selectivity of the glucose residues and the target protein, giving purified product with a high yield of recovery.
1• It can be a one step process in many cases.
2• The technique can be used for substances of low concentration.
3• Rapid separation is achieved while avoiding contamination.
4. Unlike Gel filtration chromatography and ion-exchange chromatography, affinity chromatography would be able to isolate one specific protein at a time, where other techniques will isolate proteins with similar characteristics.
• The interaction of proteins of interest and ligand has to be determined carefully. This process required expensive materials, time, and small amount of protein that can be processed at once.
Biochemistry, Berg, 6th edition, ISBN 0-7167-8724-5