Affinity chromatography

Affinity chromatography is a sample purification technique,used primarily for biological molecules such as proteins.
1.Principle
2.Theory
3.Instrumentation
4. Applications

1. AFFINITY CHROMATOGRAPHY Prepared BY: REKHA T N M Pharma DEPT . OF PHARMACEUTICAL ANALYSIS SACCP 1

2. CONTENT History Introduction Principle Theory Procedure Application Advantages Disadvantages Reference 2

3. AFFINITY HISTORY 1930s, first developed by A. Wilhelm Tiselius-a Swedish biochemist, won the Nobel prize in 1948. Used to study enzymes and other proteins. Relies on the affinity of various biochemical compounds with specific properties. 3

4. INTRODUCTION Chromatography is a physical method of separation in which the components to be separated are distributed between two phases i.e., stationary phase and mobile phase which moves in a definite direction. Affinity chromatography is 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 ligand, which is immobilized on a support . If a solution of a mixture of proteins is passed through the chromatographic column one of the proteins binds to the ligand based on the specificity an high affinity (they fit together like a lock and key). 4

5. The other proteins in the solution wash through the column because they were not able to bind to the ligand. ligand bead target proteins Free ligand Elute the bound target proteins by introducing the free ligand 5

6. PRINCIPLE A ffinity 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. 6

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8. T he 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 elute. 8

9. THEORY Specificity of affinity chromatography : Specificity is based on three aspects o f affinity Matrix Spacer arm Ligand. 9

10. MATRIX The matrix simply provides a structure to increase the surface area to which the molecule can bind. Amino, hydroxyl and carbonyl groups located with the matrix serve as ligand binding sites. Matrix are made up of agarose and other polysaccharides. For having an effective matrix, it must have a certain characters : It must be chemically and mechanically stable . It must be insoluble in solvents and buffers employed in the process. It must be easily coupled to a ligand or spacer arm onto which the ligand can be attached . 10

11. It must exhibit good flow properties and have a relatively large surface area for attachment. Types of matrix used : Cellulose: used for DNA affinity chromatography Agarose: DNA or proteins Tris-acryl: It is having higher separation ability It has small particle size 40-80 micrometer . 11

12. SPACER ARM T he stationary phase is typically a gel matrix, to prevent steric interference or overlap during the binding process of the target molecule to the ligand, an inhibitor containing a hydrocarbon chain is first attached to the agarose bead (solid support). T his inhibitor with a hydrocarbon chain is commonly known as the spacer between the agarose bead and the target molecule. 12

13. LIGAND The ligand binds only to the desired molecule within the solution. It attaches to the matrix which is made up of an inert substance. It should only interact with the desired molecule and form a temporary bond. The ligand/complex molecule will remain in the column, eluting everything else off. The ligand/molecule complex dissociates by changing the pH. The ligand can be selected only after the nature of the macromolecule to be isolated is known. 13

14. 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 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. 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 substances. It must have chemically modifiable groups that allow it to be attached to the matrix without destroying binding activity. 14

15. Immobilization of ligands: Immobilization of affinity ligand is 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. These multiple attachment sites causes the ligand to become denatured or distorted, multi site attachment can lead to reduced binding affinity. 15

16. Covalent immobilization : Covalent immobilization is one of the most common ways of attaching an affinity ligand molecule to a solid support material. There are 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. 16

17. Adsorption of affinity ligands; Adsorption can be either nonspecific or specific. Non specific adsorption : in Non specific adsorption the affinity ligand simply adsorbs to the surface of the support material and is a result of 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 for the adsorption of antibodies in and/or hydrophobic interactions. 17

18. Attachment of ligand to matrix. Several procedures have been developed for the covalent attachment of the ligand to the stationary phase. All procedures for gel modifications proceed in two separate chemical steps: Activation of the functional group on the matrix. Joining of the ligand to the functional group on the matrix. 18

19. Examples : Antigen Antibody Antibody Antigen Substrate Enzyme DNA Histon Hormone Binding protein/receptor. 19

20. Procedure Affinity chromatography technique involves three main steps: Bind Wash elute 20

21. Step-1: Attach ligand to column matrix . Binding of the selected ligand to column matrix requires a covalent bond to be formed between them. Most ligands are attached first to spacer arms and then bonded to matrix. The ligand matrix gel is then loaded into an elution column. 21

22. Once the column has been prepared, the mixture containing isolate is poured into the elution column. Gravity pulls the solution through the gel, because most of the proteins do not bind to the ligand matrix complex. When ligand is recognized substrate passes through the gel, it binds to the ligand matrix complex, halting its passage through the gel. Some of the impurities flow through the gel due to its gravity, but most remain unbound in the gel column. Step-2: Load protein mixture onto column . 22

23. Step-3: Proteins bind to ligand . In order to remove these unbound impurities, a wash of extreme pH, salt concentration, or temperature is run through the gel . It is important to use a strong wash so that all the impurities are removed. Once the impurities are washed out, the only remaining part of the protein mixture should be the desired isolates. 23

24. Step-4: Wash column to remove unwanted material In ally to collect isolate, which is still bound to the ligand matrix in the gel, a stronger second wash is run through the column. 24

25. Step-5: Wash of proteins that bind loosely This second wash relies on the reversible binding properties of the ligand, which allows the bound protein to dissociate from its ligand in the presence of this stronger wash . 25

26. Step-6: Elute proteins that bind tightly to ligand and collect purified protein of interest. The protein is then free to run through the gel and be collected. 26

27. A pplication Immunoglobulin purification (antibody immobilization ) Used to purify antibody against a specific antigen Ex: Immunoglobulin's 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’- carbonyl di-imidazole, tresylchloride 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. 27

28. 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. 28

29. 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. 29

30. His-tagged protein purification H istidine-tagged recombinant protein purification using Immobilized metal ion chromatography(IMAC) . Ni2+ Sepharose resins are precharged with nickel ions (Ni2+) metal ion affinity chromatography. Ni2 + Sepharose excel is especially suitable for purification of histidine tagged proteins secreted into eukaryotic cell culture supernatants . IMAC resins charged with Ni2+ and Co2+ 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 ( Cu2+) or zinc (Zn2+). In these cases, uncharged IMAC resins can be conveniently charged with the metal ion of your choice. 30

31. 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. 31

32. 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). 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. 32

33. 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/N acetyl galactosamine , N acetyl glucosamine, fructose, and N-acetyl neuraminic 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. 33

34. 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. IMAC columns clarify plasmid DNA from bacterial alkaline lysates, purify a ribozyme, and remove primers and other contaminants from PCR reactions. 34

35. 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 . I nitial step of reversed phase separation involves equilibration of the column under suitable conditions (pH, ionic strength and polarity. 35

36. 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. 36

37. Advantages Extremely high specificity High degree of purity can be obtained The process is very reproducible The binding sites of biological molecules can be simply investigated. 37

38. Disadvantages Expensive ligands Leakage of ligand Degradation of the solid support Limited lifetime Non-specific adsorption 38

39. References Cuatrecasas P. Protein purification by affinity chromatography: derivatizations of agarose and polyacrylamide beads. Journal of Biological Chemistry. 1970 Jun 25;245(12):3059-65 . Uhlén M. Affinity as a tool in life science. Biotechniques. 2008 Apr;44(5 ):649-54 . Hage DS. Affinity chromatography: a review of clinical applications. Clinical chemistry. 1999 May 1;45(5):593-615. 39

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