Presented by:Samya SayantanId: 121-29-381Batch: 7th

Sec: ADepartment of PharmacyDaffodil International University

Introduction:Affinity chromatography is principally based on the molecular

recognition of a target molecule by a molecule bound to column.

Affinity purification involves 3 main steps:

a. Incubation of a crude sample with the affinity support to allow the target molecule in the sample to bind to the immobilized ligand.

b. Washing away non-bound sample components from the support.

c. Elution (dissociation and recovery) of the target molecule from the immobilized ligand by altering the buffer conditions so that the binding interaction no longer occurs.

Definition:Affinity Chromatography is a separation method based

on a specific binding interaction between an immobilized ligand and it’s binding partner.

Example: Antigen AntibodyAntibody AntigenSubstrate EnzymeDNA HistonHormone Binding Protein/Receptor

Principle: The stationary phase is typically as gel matrix (often

agarose). The molecule of interest has a known and defined

property. The process is an entrapment in which the target

molecules becomes trapped on stationary phase. The stationary phase can then be removed from

the mixture, washed and then target molecules is released from the entrapment.

Chromatographic Media A matrix in its use here is a substance, usually in

bead form to which a specific ligand is covalently bound.

In order to for the matrix to be effective it must have certain characters:

It must be insoluble in solvents and buffers employed in the process.

It must be chemically and mechanically stable. It must be easily coupled to a ligand or spacer

arm onto which the ligand can be attached. It must exhibit good flow properties and have a

relatively large surface area for attachment

Immobilized Ligand: The ligand can be selected only after the nature of the

macromolecule to be isolated is known. When a hormone receptor protein is to be purified by affinity

chromatography, the hormone itself is an ideal candidate for the ligand.

For antibody isolation, an antigen or hapten may be used as ligand.

If an enzyme is to be purified, a substrate analog, inhibitor, cofactor, or effectors may be used as a the immobilized ligand.

Attachment of Ligand:Several procedures have been developed for the

covalent attachment of the ligand to the stationary phase.all procedures for gel modification proceed in two separate chemical steps:

Activation of the functional groups on the matrix and

Joining of the ligand to the functional group on the matrix.

Materials: A bead matrix. A ligand. A solution containing the substrate to be isolated a

wash to elute the non-bound impurities in the solution.

A final wash to elute the bound substrate from its ligand.

The bead matrix is an agarose gel loaded into an elution column. Sepharose is the most widely used matrix, because the hydroxyl groups on the sugar residues can be easily manipulated to accept a ligand.

Procedure: Binding of the selected ligand to the matrix requires

that a covalent bond be formed between the two. Most ligands are attached first to spacer arms which are then bonded to the matrix. The ligand-matrix gel is then loaded into an elution column.

Once the column has been prepared, the mixture containing your favorite isolate is poured into the elution column. Once in the column, gravity pulls the solution through the gel, because most of the proteins do not bind to the ligand-matrix complex. However, when the ligand's 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 gravity, but most remain, unbound, in the gel column.

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, but it is also just as crucial that the wash be not so strong that it removes the bound isolates. Once the impurities are washed-out, the only remaining part of the protein mixture should be the desired isolates.

Finally to collect the isolate which is still bound to the ligand-matrix in the gel, a stronger second wash is run through the column. 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. The protein is then free to run through the gel and be collected.

Application: Purifying a protein based on histidine –

nickel interaction:

A major problem in the field of molecular biology involves the expression and purification of a protein of interest, for example the human adenovirus serotype, Ad5. This protein has been commonly used as a vector for gene therapy, due to its efficient gene delivery and application in a diverse range of cells. A simple one step metal affinity purification system, utilizing the His tag which binds to Ni-NTA, was used to isolate the Ad5, with greater than 95% purity. The isolated protein was evaluated via a receptor-binding assay in Hela cells.

Purifying a protein based on inherent ligant-binding specificity:

There are a few proteins that have natural affinity to bind metal ions. This inherent metal ion binding has been utilized as a way to purify these proteins using metal immobilized affinity columns. For example, a red fluorescent protein, DsRed, has a binding affinity for copper ions. On the basis of this a purification method for DsRed was developed using copper immobilized column. A metal chelating ligand attached to beads was used in this study to immobilize copper ions on beads. The crude protein was passed through the copper immobilized beads. DsRed bound to the beads whereas other interfering proteins did not bind to the beads and hence were removed. DsRed was eluted using a competitive ligand imidazole that binds to copper ions. This is a simpler approach of protein purification based on their inherent metal ions affinity.

Study of drug interaction:

The quantitative characterization of interactions between targets and ligands is an integral part of the drug discovery process. These interactions are often defined by the association constant of a drug-protein interaction. The association constants, for a number of HSA-binding drugs based upon their retention factors. They prepared an immobilized HSA column by passing known concentrations of the drugs through the column saturation plots were generated which were used to calculate retention factors and association constants. This system offers advantages over the more common methods of evaluating association constants such as fast response times ease of automation, and additionally the ability to distinguish chiral compounds simultaneously.

Advantages

Extremely high specificity High degrees of purity can be

obtained The process is very reproducible The binding sites of biological

molecules can be simply investigated

Disadvantages

Expensive ligands Leakage of ligand Degradation of the solid support Limited lifetime Non-specific adsorption Relatively low productivity