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Affinity Chromatography

1. AFFinity chromatography Submitted by, NAVAMI S S 1 st yr Biochemistry Dept.of Life Sciences

2. Introduction Affinity chromatography is a powerful technique of purifying proteins. Biological macromolecules, such as enzymes and other proteins, interact with other molecules with high specificity through several different types of bonds and interaction. Such interactions include hydrogen bonding, ionic interaction, disulfide bridges, hydrophobic interaction, and more. The high selectivity of affinity chromatography is caused by allowing the desired molecule to interact with the stationary phase and be bound within the column in order to be separated from the undesired material which will not interact and elute first. The molecules no longer needed are first washed away with a buffer while the desired proteins are let go in the presence of the eluting solvent (or higher salt concentration).

3. Affinity Chromatography Chromatography is an important biophysical technique that enables the separation, identification, and purification of the components of a mixture for qualitative and quantitative analysis.It is a separation technique in which a mobile phase carrying a mixture is caused to move in contact with a selectively absorbent stationary phase.
Affinity chromatography is a type of liquid chromatography for the separation, purification or specific analysis of sample components.It utilizes the reversible biological interaction or molecular recognition called affinity which refers to the attracting force exerted in different degrees between atoms which cause them to remain in combination. Example: Enzyme with an inhibitor, antigen with an antibody, etc. × It was discovered by Pedro Cuatrecasas and Meir Wilcheck .

4. Principle The stationary phase consists of a support medium, on which the substrate (ligand) is bound covalently, in such a way that the reactive groups that are essential for binding of the target molecule are exposed. As the crude mixture of the substances is passed through the chromatography column, substances with binding site for the immobilized substrate bind to the stationary phase, while all other substances is eluted in the void volume of the column.

5. After all the other proteins have been removed and bound, it is now possible to bind the enzyme(s) are able to be removed in a variety of ways: 1. by increasing the ionic strength of buffer e.g. By introducing the sodium chloride gradient which weakens the interactions that the enzyme has with its immobilised substrate.
2. by altering the pH in the buffer. This is thereby lessening the interaction with the substrate and enzyme.
3. by adding a significant amount of substrate (or an analogue of the substrate) to the buffer for elution, to ensure that there is a competing between the immobilised and free substrates for the enzyme protein.

6. Components of affinity chromatography Matrix The matrix acts as an inert structure that a ligand may be connected directly or indirectly. The most efficient matrix materials are polyacrylamide and agarose. It is distinguished by its unique properties like; Doesn’t itself absorb molecules in a substantial quantity.
Ligands should be coupled without altering the binding properties.
Stability in a variety of conditions for experiments, such as both low and high pH detergent, as well as dissociating conditions.

7. 2. Spacer arm It prevents the ligand from attaching to the matrix, which could interfere with its ability to bond to macromolecules. The optimal length is 6-10 carbon atoms or equivalent. Most commonly, it is utilized for smaller immobilized ligands. Examples of Spacer arms are 1,6-diamino Hexane and 6-amino Hexanoic Acid.

8. 3.Ligand The ligand is a molecule which binds reversibly with an individual chemical or group of molecules, making it possible to purify the sample using affinity chromatography. The choice of a specific ligand to be used for affinity chromatography can be influenced by two aspects: The ligand has to show an irreversible and specific binding affinity for the targeted substance(s)
It should possess chemically modifiable groups that permit it to be bonded to the matrix without damaging the binding function.

9. Any element can be utilized as a ligand for purification of the binding partner of its choice. A few of the most common biological interactions often used in affinity chromatography are as follows: Enzyme used for substrate analog and inhibitors, as well as cofactor.
Antibody used for antigen, virus, cell.
Lectin is a polysaccharide glycoprotein cell surface receptor cell.
Nucleic acid is used to complement base sequences, histones nucleic acid polymerase and nucleic acid-binding protein.
Hormone vitamin, used to receptors, carriers protein.
Glutathione used for glutathione-S- transferase or GST fusion proteins.
Metal ions that are used in Poly (His) fusion proteins and native proteins that contain histidine, cysteine or tryptophan residues on their surface.

10. Steps of Affinity Chromatography Step: 1 Attach ligand to column matrix The binding of the ligand with the matrix requires that a covalent bond is created between both. This is accomplished through derivatization of the sugar-based” hydroxyl groups. The substrate may not be able to access the active site of the ligand, in the event that it is hidden inside the ligand. The majority of ligands are connected to spacer arms that are then attached onto the matrix. The matrix-ligand gel is loaded into an column of elution.

11. Step 2: Load Protein Mixture onto the Column After the column is made, the mix containing isolate is then poured into the column for elution. Gravity pulls the solution into the gel because most of the proteins don’t attach to the ligand-matrix. If ligand that is recognized as substrate moves through the gel it bonds to the ligand-matrix complex, stopping its flow within the gel. Certain impurities pass through the gel because of gravity, however the majority remain unbound in the gel column. Step 3: Proteins Binds to the ligand To remove these impurities which are not bound, the wash must be of high pH or salt concentration or temperature is passed across the gel. It is crucial to make the most powerful wash possible to ensure all impurities are eliminated. After the impurities have been removed then the only thing left of the protein mixture must be the specific isolates.

12. Step 4: Wash the column to remove the unwanted Materials To finally collect an isolate that is attached to the ligand matrix in the gel second wash is run over the column. Step 5: Wash off the Proteins that are loosely bind The second wash is based on the reversible properties of binding of the ligand. This lets the bound protein separate from its ligand in the presence of the more powerful wash. Step 6: Elute proteins that are tightly bound to ligand and collect purified protein and interest The protein then has the freedom to pass through the gel before being removed.

13. Elution methods of Affinity Chromatography 1.pH elution A variation in pH alters the amount of ionization by charged groups on the ligand as well as proteins bound. The alteration could affect site of binding directly decreasing its affinity or trigger an indirect change in affinity due to modifications in the conformation. A gradual decrease by a step in pH can be the most popular method of eluting bound substances. Chemical stabilities of the matrix, the ligand, and the target protein determines the limits of pH which can be used. If a low pH is required, then collect the fractions into neutralization buffers like 1 M Tris-HCl .pH 9 (60-200 mg ul for each ml fraction eluted) to bring the fraction back to an equilibrium pH. The column should be returned in the neutral pH immediately .

14. 2. Ionic strength elution The exact mechanism behind elimination through changes in the strength of ionic depends on the specific relationship between the ligand and the target protein. This is a moderate elimination using a buffer that has higher Ionic strength (usually NaCl ), applied in a linear manner. 3. Competitive elution They are typically used to distinguish substances in an individual medium or when the attraction of the target protein interactions is quite high. The eluting agent is competing with the protein of interest or for binding to the binding ligand. Substances can be eluted by the concentration gradient of one Eluent, or by pulse elimination. In the case of competitive elution the concentration of the competing compound should be equal to that of the ligand that is coupled. If, however, the competing compound is able to bind more easily than the ligand targeted molecule, then use an amount that is ten times greater than the ligand.

15. 4. Reduced polarity of eluent Conditions are employed to reduce the polarity of the eluent and allow for elution but not inactivating the substances that are eluted. Dioxane (up to 10 10%) as well as Ethylene glycol (up to 50 percent) are common to this kind of liquid eluent. 5. Chaotropic eluents If other methods of elution fail the deforming buffers which alter the protein’s structure can be employed. e.g. Chaotropic agents like the guanidine hydrochloride and urea. Avoid chaotropes whenever possible as they can cause to cause denature of the protein eluted.

16. Types of Affinity Chromatography 1.Lectin Affinity Chromatography Purification of glycoproteins, specifically the membrane-receptor proteins.
Lectins are a class of proteins made by animals and plants that are able to bind glycoproteins and carbohydrates.
Useful to separate cells into different types, making use of saccharide component of their outer membranes.
The most commonly used lectins include: ConcanavalinA Soyabean lectin, etc..

17. 2. Immuno Affinity chromatography It is used in the isolation and elimination of a variety of proteins such as antigens, membrane proteins that are of viral origin.
It is used to purify antibodies.
Ligands used include the Protein A as well as Protein G. 3. Metal Chelate Chromatography Special type of chromatography which immobilised metal ions such as Cu2+ Zn2+ , Mn2+, Ni2+ etc. are employed.
It is used to purify proteins that contain imidazole groups or indole groups.
Commonly metal ions are immobilised by attachment to an imino-diacetate or tris ( carboxymethyl ) ethylenediamine substituted agarose.

18. 4. Dye Ligand Chromatography Utilizes a variety of triazine dyes for ligands.
The most popular color is Cibracron Blue F3G-A.
It is used to purify interferons and lipoproteins as well as factors that cause coagulation, etc. 5. Covalent chromatography Specially designed to separate proteins containing thiol The most frequently used ligand an adiquate 2′-pyridyl group
It is used to purify many proteins, however its application is restricted due to its price and difficult regeneration.

19. Types of affinity media used in Affinity Chromatography A variety of affinity media are available to serve a range of applications. Briefly, they are (generalized) activated/functionalized that work as a functional spacer, support matrix, and eliminates handling of toxic reagents. Amino acid media: It is used in conjunction with a variety of proteins from serum enzymes, and peptides in addition to dsDNA and rRNA . Avidin biotin media: Avidin biotin media is used to purify the process of biotin/ avidin , as well as their derivatives.
Carbohydrate bonding is most often used with glycoproteins or any other carbohydrate-containing substance; carbohydrate is used with lectins, glycoproteins, or any other carbohydrate metabolite protein.

20. The dye ligand medium is nonspecific however it mimics biological substrates as well as proteins. Hydrophobic interaction medium are frequently employed to attack free carboxyl groups and proteins. Immunoaffinity media uses antigens’ as well as antibodies that have high specificity to differentiate immobilized metal affinity chromatography . It uses interactions between proteins and metal ions (usually specifically labeled ) to separate. Nucleotide/coenzyme which helps separate dehydrogenases, kinases and transaminases. Speciality media are made for specific classes or types of co enzyme. This kind of media can only function to isolate a particular type of protein, or coenzyme.

21. Weak affinity chromatography WAC is a weak affinity technique. (WAC) is an affinity chromatography technique used for testing affinity for drug development. It is an affinity-based technique for liquid chromatography which separates chemical compounds according to their weak affinities with the immobilized object.
The greater affinity a compound has with the target the longer it will remain within the separator and this is measured as a longer retention time.
The measurement of affinity and rank of affinity are accomplished by processing the retention times of the compounds being studied.

22. Affinity chromatography is a part of a wider set of chemoproteomics techniques for the purpose of identifying drug targets.
The WAC technology has been demonstrated against various protein targets , including proteases chaperones, kinases, along with protein-protein interaction (PPI) specific targets. WAC has been proven to be more efficient than traditional methods for screening based on fragments.

23. Application of Affinity Chromatography It is used to isolate and purification of all biological macromolecules.
It is used to purify nucleic acid, antibodies, enzymes etc To decrease the amount of substance present in a mix.
Utilized for Genetic Engineering for nucleic acid purification
Utilized for the Production of Vaccines – antibody purification from blood serum
It is used for Basic Metabolic Research such as the purification of enzymes or proteins from cells free extracts.
Affinity chromatography also serves to get rid of particular contaminants, like that of Benzamidine . Sepharose (tm) 6 Fast Flow removes serine proteases like the Factor Xa and thrombin.

24. Antibodies can be immobilized by both covalent and adsorption methods. Random covalent immobilization methods generally link antibodies to the solid support via their free amine groups using cyanogen bromide, N- hydroxysuccinimide , N,N’- carbonyldiimidazole , tresyl chloride, or tosyl chloride. Alternatively, free amine groups can react with aldehyde or free epoxy groups on an activated support. As these are random immobilization methods, the antibody binding sites may be blocked due to improper orientation, multi-site attachment or steric hindrance. Site-specific covalent immobilization of antibodies can be achieved by converting the carbohydrate residues located in the Fc region of the antibody to produce aldehyde residues which can react with amine or hydrazide supports. 1. IMMUNOGLOBULINS PURIFICATION (antibody immobilization)

25. 2. Recombinant tagged proteins Purification of proteins can be easier and simpler if the protein of interest is tagged with a known sequence commonly referred to as a tag. This tag can range from a short sequence of amino acids to entire domains or even whole proteins. Tags can act both as a marker for protein expression and to help facilitate protein purification. 3. PROTEIN A, G, & L PURIFICATION Proteins A, G, and L are native or recombinant proteins of microbial origin which bind specifically to immunoglobulins including immunoglobulin G (IgG). IgG represents 80% of serum immunoglobulins. The most popular matrixes or supports for affinity applications which utilize protein A, G, or L is beaded agarose.

26. 3a. GST tagged purification Separation and purifcation of GST-tagged proteins is possible since the GST tag is capable of binding its substrate, glutathione (tripeptide, Glu-Cys-Gly ). When glutathione is reduced (GSH), it can be immobilized onto a solid support through its sulfhydryl group. This property can be used to crosslink glutathione with agarose beads and, thus, can be used to capture pure GST or GST-tagged proteins via the enzyme-substrate binding reaction. Binding is most efficient near neutral physiological conditions (pH 7.5) using Tris saline buffer and mild conditions to preserve the structure and enzymatic function of GST. As a result of the potential for permanent denaturation, denaturing elution conditions are not compatible with GST purification. In addition, upon denaturation or reduction, the structure of the GST fusion tag often degrades.

27. 3b. His-tagged protein purification Recombinant proteins which have histidine tags can be purified using immobilized metal ion chromatography (IMAC). The His-tag can be placed on either the N- or C-terminus. Optimal binding and, therefore, purification efficiency is achieved when the His-tag is freely accessible to metal ion support. 4. BIOTIN & BIOTINYLATED MOLECULES PURIFICATION If a biotin tag can be incorporated into a biomolecule, it can be used to purify the biomolecule using a streptavidin or avidin affinity support.

28. 6.Affinity purification Of albumin &macroglobulin Contamination Affinity purification is a helpful tool for cleaning up and removing excess albumin and α2- macroglobulin contamination from samples since these components can mask or interfere with subsequent steps of analysis 5. ENZYME ISOLATION There are a number of areas related to affinity chromatography that have also been of great interest in pharmaceutical and biomedical analysis. One such area is in the use of affinity columns for enzyme isolation. This approach may use affinity ligands that are substrates, inhibitors, cofactors, or proteins that are associated with the biochemical pathways of the target enzyme

29. 7. Protein and Peptide Separations Affinity chromatography has a wide range of applications for protein purification, such as immunoaffinity , purification of immunoglobulins, purification of glycoproteins, DNA-binding proteins, receptor proteins, enzymes, cell isolation, and nucleotide isolation. Affinity chromatography is a specific purification technology based on biological function or individual chemical structure. The application of this technique is in separation of active biomolecules from denaturated or functionally different forms in the isolation of pure proteins present at low concentration and also for removing specific contaminants.

30. 8. Investigating protein-protein interactions Affinity chromatography provides one important method for identifying and characterizing the intermolecular interactions. When investigating protein-protein interactions, affinity chromatography typically involves linking one protein to an insoluble matrix and then incubating that matrix with a solution containing possible binding partners. This solution could be as simple as homogenous solution containing a single recombinant protein or complex. After incubating the immobilized substrate protein with its potential binding partner(s) and washing away material associated non specifically the binding partner are the eluted and detected by any variety of methods from chromatographic detection

31. Advantages of Affinity Chromatography Extremely high-specificity
The purest of levels can be achieved
The process is highly reproducible.
The binding sites of biological molecules could be investigated by simply looking at the binding sites of biological molecules.
Single-step purification.
The matrix is reusable in a short time and is solid that is easy to clean and dried.
Provide purified products with high yield.
Affinity chromatography may also be used to get rid of particular contaminants, for instance proteases.

32. Disadvantages of Affinity Chromatography Expensive ligands
Leakage of the ligand
Degradation of the solid support
Relatively low productivity
Non-specific adsorption can not be totally eliminated, it can only be minimized.
The limited life span and the high cost for immobilized ligands.
Proteins are denatured when the necessary pH is not maintained.