Affinity Chromatography - KSUfac.ksu.edu.sa/sites/default/files/bch_332_lecture_13.pdf ·...

Affinity ChromatographyBCH 332 Lecture 13

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• Based on the binding affinity of a protein.

• The beads in the column have a covalently attached chemical group.

• A protein with affinity for this particular chemical group will bind to the beads in the column, and its migration will be retarded as a result

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• The matrix simply provides a 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.

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• 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.

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Types of Matrix

• Cellulose : used for DNA affinity chromatography.

• Polyacrylamide : It exist in gel & in form of beads.

• Agarose

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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 buffer.

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• Antigen Antibody

• Substrate Enzyme

• DNA Histone

• Hormone Receptor/Hormone binding protein

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• Enzymes may be purified by affinity chromatography using:

• immobilized substrates,

• products,

• coenzymes,

• or inhibitors.

• Elution:

1-by competition with soluble ligand

2-or, less selectively, by disrupting protein-ligand interactions using:

• urea,

• guanidine hydrochloride,

• mildly acidic pH,

• or high salt concentrations.

Common elution buffer systems for protein affinity purification

• These conditions apply primarily to protein-protein binding interactions, such as between an antibody and its peptide antigen.

• Elution buffers for binding interactions between other kinds of molecules may be quite different.

• Stationary phase matrices available commercially contain ligands such as NAD+ or ATP analogs.

• The most powerful and widely applicable affinity matrices:

• modified recombinant proteins. These include a Ni2+ matrix that binds proteins with an attached polyhistidine “tag”.

• glutathione matrix that binds a recombinant protein linked to glutathione S-transferase.

Greater than 1000-fold purification of a specific protein

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Principle

• Inject a sample into an initially equilibrated affinity chromatography column.

• Only the substances with affinity for the ligand are retained in the column.

• Other substances with no affinity for the ligand are eluted from the column.

• The substances retained in the column can be eluted from the column by changing pH or salt or organic solvent concentration of the eluent.

• Affinity chromatography is widely used as a means of separation and purification.

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Applications

• Purify and concentrate a substance from a mixture into a buffering solution.

• Reduce the amount of a substance in a mixture.

• Purify and concentrate an enzyme solution.

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• Used in Genetic Engineering - nucleic acid purification.

• Production of Vaccines - antibody purification from blood serum.

• Basic Research - protein or enzyme purification from cell free extracts

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Industrial Applications

• Affinity chromatography is widely used in the pharmaceutical industry to purify and extract molecules of interest from complex mixtures.

• These molecules can be enzymes, proteins or amino acids, but other biological species can be selectively retained.

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Clinical Applications

• Hyper-lipidemia : here the sample is made to pass through column containing antibody & plasma LDL so, it can easily be separated out by eluting with glycine hydrochloride buffer (pH 3).

• Others : Pregnancy test, Allergy test, Immuno assay, Kinetic studies, Qualitative measurement of substrate.

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Advantages

1. Extremely high specificity

2. High degrees of purity can be obtained

3. The process is very reproducible

4. The binding sites of biological molecules can be simply investigated.

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Disadvantages

1. Expensive ligands

2. Leakage of ligand

3. Degradation of the solid support

4. Limited lifetime

5. Non-specific adsorption

6. Relatively low productivity

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Fusion tag protein purification

• Proteins are expressed recombinantly,

• Fusion tags could be added

• Most common fusion tags is a short string of six to nine histidine residues (known as the 6xHis or polyHis tag), which will bind to metal ions such as nickel or cobalt.

• Other fusion tags include HA, Myc, FLAG

• These other tags are called epitope tags because they require specific antibodies (e.g., immobilized anti-HA antibody) for purification.