affinity chromatography

affinity chromatography

1. AFFINITY CHROMATOGRAPHY 1 Presented by: Ayesha samreen 1 st year M.Pharm (Pharmaceutics )

2. HISTORY 1930s, first developed by Arne Wilhelm Tiselius, won the Nobel Prize in 1948 Used to study enzymes and other proteins Relies on the affinity of various biochemical compounds with specific properties ex) enzymes for their substrates antibodies for their antigens 2

3. INTRODUCTION Affinity chromatography is essentially a sample purification technique, used primarily for biological molecules such as proteins. It is a method of separating a mixture of proteins or nucleic acids (molecules) by specific interactions of those molecules with a component known as a ligand, which is immobilized on a support. If a solution of, say, a mixture of proteins is passed over (through) the column, one of the proteins binds to the ligand on the basis of specificity and high affinity (they fit together like a lock and key). The other proteins in the solution wash through the column because they were not able to bind to the ligand. 3

4. PRINCIPLE 4 Affinity chromatography is one of the most diverse and powerful chromatographic methods for purification of a specific molecule or a group of molecules from complex mixtures It is based on highly specific biological interactions between two molecules such as interactions between enzyme and substrate, receptor and ligand, or antibody and antigen. These interactions which are typically reversible are used for purification by placing one of the interacting molecules referred to as affinity ligand onto a solid matrix to create a stationary phase while a target molecule is in the mobile phase. Many of the commonly used ligands coupled to affinity matrices are now commercially available and are ready to use .

5. EXAMPLES Antigen Antibody Substrate Enzyme 5

6. The Sample is injected into the equilibrated affinity chromatography column Only the substance with affinity for the ligand are retained on the column The substance with no affinity to the ligand will elute off The substances retained in the column can be eluted off by changing the pH of salt or organic solvent concentration of the eluent 6

7. Specificity of Affinity Chromatography Specificity is based on three aspect of affinity Matrix : for ligand attachment . Matrix should be chemically and physically inert. Spacer arm : used to improve binding between ligand and target molecule by overcoming any effects of steric hindrance. Ligand : molecule that binds reversibly to a specific target molecule(site of interaction ). 7

8. Matrix (solid support) The matrix simply provides a basic structure to increase the surface area to which the molecule can bind. The matrix must be activated for the ligand to bind to it but still able to retain it’s own activation towards the target molecule. 8

9. Matrix Amino, hydroxyl, carbonyl and thio groups located with the matrix serve as ligand binding sites Matrix are made up of agarose and other polysaccharides The matrix also must be able to withstand the decontamination process of rinsing with sodium hydroxide or urea 9

10. Solid support: a. carbohydrate polymers : ex, agarose, cellulose, dextran. Commercially available agarose in 1-12% is usually adequate. cross linking with divinylsulfone(DVS) or 1,4-butanediol diglycidyl ether increases both the chemical and thermal stability of the gel but may diminish the binding capacity. DVS increases the gel’s rigidity. Cross linked agarose tolerates any solvents except strong acids and oxidizers but may be damaged by some rare enzymes and must not be frozen or dried. Agarose gel activates complement system in human blood and must never be used in vivo ( e.g., for affinity removing of toxins from blood). 10

11. Dextran : it consists of two types of gels a. sephadex b. sephacryl Sephacryl are used in the chromatography of biomolecules. Sephacryl S-1000 is especially suitable for affinity chromatography of very large molecules. At low pH Sephacryl S-200 adsorbs proteins. Sephadex is used mainly as a glucose polymer (example for the purification of lectins ) it is mechanically to weak or has insufficient porosity . It can be activated in aqueous media with non-cross-linking reagents. 11

12. B. Synthetic polymers : polyacrylamide(PAA) is synthesized by copolymerization of acrylamide and a cross linking agent. Primary disadvantage is that the gels are either soft or have small pores. PAA gel is resistant against enzymatic attacks and does not adsorb biomolecules. Hydroxyalkylmethacrylate gels: the gel is chemically and mechanically more stable than PAA gel but is more hydrophobic and thus it is inappropriate for many applications. Trisacryl: it is hydrophilic, biologically inert, rigid, and macroporous. Despite its low working volume( it contains more than 30% of solids) it is used for large scale preparations. 12

13. C. mixed gels: Polyacrylamide-agarose gels (ultrogel AcA) were developed for gel filtration and tested for affinity chromatography. 13

14. D. inorganic materials: When extreme rigidity of the support is needed , as for HPLC and large-scale applications, inorganic materials are used ( irregular porous glass or silica particles). They are soluble pH >8, although a coating of zirconium will enhance their stability. otherwise, glass or silica may be coated with an inert polymer. 14

15. E. magnetic carriers : Magnetic sorbent particles (magnogel AcA 44 and Act-magnogel AcA 44 ) are useful for batch extraction from large volumes of diluted and/or turbid solutions. 15

16. Ligand The Ligand binds only to the desired molecule within the solution The ligand attaches to the matrix which is made up of an inert substance The ligand should only interact with the desired molecule and form a temporary bond The ligand/molecule complex will remain in the column, eluting everything else off The ligand/molecule complex dissociates by changing the pH 16

17. ligand The chosen ligand must bind strongly to the molecule of interest. If the ligand can bind to more than one molecule in the sample a technique, negative affinity is performed. - this is the removal of all ligands, leaving the molecule of interest in the column. - done by adding different ligands to bind to the ligands within the column. 17

18. The selection of the ligand for affinity chromatography is influenced by two factors: the ligand must exhibit specific and reversible binding affinity for the target substance(s) and it must have chemically modifiable groups that allow it to be attached to the matrix without destroying binding activity. 18

19. Immobilization of ligand Immobilization of the affinity ligand is also very important when designing an affinity chromatography method for biomolecule purification. Activity of the affinity ligand can be affected by multi-site attachment, Multi-site attachment occurs when an affinity ligand is attached through more than one functional group on a single ligand molecule. orientation of the affinity ligand, and If these multiple attachment sites cause the affinity ligand to become denatured or distorted, multisite attachment can lead to reduced binding affinity. However, in some instances, the additional attachment sites can result in more stable ligand attachment steric hindrance. 19

20. Covalent immobilization Covalent immobilization is one of the most common ways of attaching an affinity ligand to a solid support material. There is a wide range of coupling chemistries available when considering covalent immobilization methods. Amine, sulfhydryl, hydroxyl, aldehyde, and carboxyl groups have been used to link affinity ligands. covalent attachment methods are more selective than other immobilization methods, they generally require more steps and chemical reagents onto support materials. 20

21. Adsorption of affinity ligands adsorption can be either nonspecific or specific. Nonspecific adsorption : In nonspecific adsorption the affinity ligand simply adsorbs to the surface of the support material and is a result of Coulombic interactions, hydrogen bonding and/or hydrophobic interactions. Bio specific adsorption : it is commonly performed by using avidin or streptavidin for the adsorption of biotin containing affinity ligands or protein A or protein G for the adsorption of antibodies in, and/or hydrophobic interactions. 21

22. Space arm the binding sites of the target molecule are sometimes deeply located and difficult to access due to steric hindrance, a spacer arm is often incorporated between the matrix and ligand to facilitate efficient binding and create a more effective and better binding environment . The length of these spacer arms is critical. Too short or too long arms may lead to failure of binding or even non-specific binding. In general, the spacer arms are used when coupling molecules less than 1000 Da. 22

23. Affinity purification involves 4 steps: 1. preparing the sample to load on the affinity column. 2. loading and incubation of the sample with ligand to promote binding 3. washing away the non bound components from the column 4. eluting(recovering) the bound components. 23

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25. Elution Adsorbed compounds can be washed off the column in two ways: by specific ( concurrent) or by non specific elution. Specific elution is based on direct interruption by analogs of either ligand or adsorbate in to the complex formed on the affinity resin. non specific elution is by changing the media atmosphere (e.g. changing the ionic strength, pH or polarity) If the affinity interaction is mainly hydrophobic, elution with detergent or mixed solvents( up to 50% ethylene glycol or isopropanol) is promising but if the interaction is ionic, high salt concentration or pH alteration are used. Elution from antigen-antibody complexes can be modeled on polystyrene microplates: immune complexes are dissociated without eluting the adsorbed antigen, while all parameters are controlled by ELISA technique. 25

26. pH elution : A change in pH alters the degree of ionization of charged groups on the ligand and/or the bound protein. A step decrease in pH is the most common way to elute bound substances. The chemical stability of the matrix, ligand and target protein determines the limit of pH that may be used 26

27. Ionic strength elution: The exact mechanism for elution by changes in ionic strength will depend upon the specific interaction between the ligand and target protein. This is a mild elution using a buffer with increased ionic strength (usually NaCl), applied as a line. e.g., Enzymes usually elute at a concentration of 1 M NaCl or lesser gradient or in steps. 27

28. Competitive elution : Selective eluents are often used to separate substances on a group specific medium or when the binding affinity of the ligand/target protein interaction is relatively high. The eluting agent competes either for binding to the target protein or for binding to the ligand. Substances may be eluted either by a concentration gradient of a single eluent. 28

29. . Applications of affinity chromatography 1 Immunoglobulin purification (antibody immobilization ) : Used to purify antibody against a specific antigen Ex:Immunoglobulins 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. As these are random immobilization methods, the antibody binding sites may be blocked due to improper orientation, multi-site attachment or steric hindrance. Antibodies can also be immobilized by adsorbing them onto secondary ligands. 29

30. 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. most commonly used tags are glutathione-S- transferase (GST), histidine fusion (His or poly His tag) and protein A fusion tags. Other types of fusion tags are also available including maltose-binding protein , thioredoxin, NusA ,GB1 domain for protein G 30

31. GST tagged purification The purification method is based on the high affinity of GST for glutathione. When applied to the affinity resin, GST-tagged proteins bind to the glutathione ligand, and impurities are removed by washing with binding buffer. Tagged proteins are then eluted from the chromatography resin under mild, non-denaturing conditions that preserve both protein structure and function. GST Buffer Kit contains prepared buffer concentrates for binding, washing, and elution of GST-tagged protein detection 31

32. His-tagged protein purification histidine-tagged recombinant protein purification using immobilized (IMAC ). Ni 2+ Sepharose resins are precharged with nickel ions (Ni 2+ ) metal ion affinity chromatography.   Ni 2+ Sepharose excel is especially suitable for purification of histidine-tagged proteins secreted into eukaryotic cell culture supernatants . IMAC resins charged with Ni 2+  and Co 2+  are the most commonly used methods for the purification of histidine-tagged proteins. However, in some cases, other metal ions may be more suitable, for example copper ( Cu 2+ ) or zinc (Zn 2+ ). In these cases, uncharged IMAC resins can be conveniently charged with the metal ion of your choice. 32

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34. Protein A, G, and L purification: Proteins A, G, and L are native or recombinant proteins of microbial origin which bind specifically to immunoglobulins including immunoglobulin G (IgG ). The most popular matrixes or supports for affinity applications which utilize protein A, G, or L is beaded agarose (e.g. Sepharose CL-4B; agarose cross-linked with 2,3dibromopropanol and desulphated by alkaline hydrolysis under reductive conditions), polyacrylamide, and magnetic beads. 34

35. Biotin and biotinylated molecules purification: Biotin: ( vitamin H or B7) cofactor in the metabolism of fatty acids and leucine, and in gluconeogenesis . In affinity chromatography it is often used an affinity tag due to its very strong interactions with avidin and streptavidin. One advantage of using biotin as an affinity tag is that it has a minimal effect on the activity of a large biomolecule due to its small size (244 Da). 35

36. Streptavidin is a large protein (60 kDa) that can be obtained from Streptomyces avidinii and bind biotin. Avidin is a slightly larger glycoprotein (66 kDa) with slightly stronger binding to biotin . Both avidin and streptavidin have four subunits that can each bind one biotin molecule . Due to the strong interaction between biotin and (strept)avidin, harsh elution conditions are required to disrupt the binding. 36

37. Lectin affinity chromatography: Lectins are carbohydrate binding proteins that contain two or more carbohydrate binding sites and can be classified into five groups according to their specificity to the monosaccharide. They exhibit the highest affinity for: mannose, galactose/Nacetylgalactosamine, N- acetylglucosamine , fructose, and N- acetylneuraminic acid. In this affinity technique, protein is bound to an immobilized lectin through its sugar moieties . Once the glycosylated protein is bound to the affinity support, the unbound contaminants are washed away, and the purified protein is eluted. 37

38. Nucleic acid separation using immobilized metal affinity chromatography (IMAC) The method can be used to purify compounds containing purine or pyrimidine moieties where the purine and pyrimidine moieties are shielded from interaction with the column matrix from compounds containing a non-shielded purine or pyrimidine moiety or group . Thus , double-stranded plasmid and genomic DNA, which has no low binding affinity can be easily separated from RNA or oligonucleotides which bind strongly to metal-charged chelating matrices 38

39. IMAC columns clarify plasmid DNA from bacterial alkaline lysates, purify a ribozyme, and remove primers and other contaminants from PCR reactions 39

40. Reversed phase chromatography Reversed phase chromatography is a kind of affinity interaction between a biomolecule dissolved in a solvent (mobile phase) that has some hydrophobicity (e.g. proteins, peptides, and nucleic acids) and an immobilized hydrophobic ligand (stationary phase ). When using reversed phase chromatography, the most polar macromolecules are eluted first and the most nonpolar macromolecules are eluted last: the more polar (hydrophilic) a solute is, the faster the elution and vice versa. table for separating non-volatile molecules. 40

41. initial step of reversed phase separation involves equilibration of the column under suitable conditions (pH, ionic strength and polarity. Next , sample is applied and bound to the immobilized matrix . Following this step, desorption and elution of the biomolecules is achieved by decreasing the polarity of the mobile phase (by increasing the percentage of organic modifier in the mobile phase). At the end of the separation, the mobile phase should be nearly 100% organic to ensure complete removal of all bound substances. 41

42. Reference: Affinity chromatography by GE healthcare ( http ://proteins.gelifesciences.com/~/ media/protein-purification-ib/documents/handbooks/affinity-chromatography-handbook-vol-2.pdf ) Affinity chromatography: principles and applications- InTech open ( http :// cdn.intechopen.com/pdfs/33046.pdf ) Affinity chromatography - journal homepage: www.elsevier.com/locate/ymeth . https:// www.slideshare.net/imaginarybiologist/affinity-chromatography www.google.com (images) 42