Affinity Chromatography.

Affinity ChromatographyA Bioanalytical Tool

Guided by:

Mr. S.A.Pishwikar Sir.

Asst. Professor.

Presentation By:

Mr. Arpit H. Patel.

M.Pharm 1st sem.

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Department of Quality Assurance,Department of Quality Assurance,Bharati Vidyapeeth College Of Pharmacy, Kolhapur.Bharati Vidyapeeth College Of Pharmacy, Kolhapur.

Outlines of the Seminar:

Abbrevations.

Introduction.

Principle.

Steps of the technique.

Applications in Bioanalysis.

Recent advances.

Conclusion.

References.2

Abreviations:

GST – Glutathione S- transferace.

HIS – Polyhistidine/hexahistidine.

EDTA – Ethylene diamine tetra acetic acid.

IgG – Immunoglobulin G.

β2 AR- β2 Adrenoreceptors.

mAChR – Muscarinic acetycholine

receptors.

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Introduction:

Gel filtration Hydrophobic interaction Ion exchange Affinity

Various Biomolecule Purification Techniques.Fig No. 1.

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Various Purification Techniques.

Affinity chromatography.

• Unique purification technique.

• Separates active biomolecules.

Defination.

Liquid chromatographic technique.

biological interaction.

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Matrix Spacer arm Ligand Molecule of interest

Design of affinity chormatography model.Fig No. 2

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Components of affinity chromatography.

Principle:

Liquid adsorption chromatography.

Reversible adsorption.

Biospecific interaction.

◦Electrostatic interactions.

◦Van der Waals' forces.

◦Hydrogen bonding.

Ligand is immobilised to an matrix.

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Steps of Affinity Chromatography:

Three steps:

a) Equilibration.

b) Sample application and wash.

c) Elution.

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a) Equilibration.

• Desired conditions.

• A matrix.

• A ligand.

• Covalant coupling.

• Retention of affinity.

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Properties of Matrix.

◦An inert support.

◦Good flow properties.

◦An open pore structure.

◦Stability.

◦Degree of crosslinking.

◦ E.g. Sepharose.

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Selection of Ligand :

◦Binds reversibly.

◦Chemically compatability towards

matrix.

◦Attachment site.

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Selection of Spacer Arm:

◦Serves as a bridge.

◦Depends on situation.

◦Maximizes target binding.

The spacer arm creates a more effective environment for binding.

Fig No. 3.

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b)Sample applications and

wash.

• Mixture is poured.

• Target molecules gets binded.

• Unbound molecules are washed out .

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c) Elution

• Recovery.

• Specifically - competitive ligand.

• Non-specifically.

o buffer composition.

o Buffer pH.

o Ionic strength.

o Polarity.

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a) Equilibration.

b) Sample application and wash.

c) Elution.

Graph No. 1.

the level of complexity

qualitative information

quantitative information

total column performance

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Time

Some typical biological interactions:Glutathione - glutathione-S-transferase or

GST fusion proteins.

Metal ions - Poly (His) fusion proteins, native proteins with histidine, cysteine and/or tryptophan residues on their surfaces.

Nucleic acid - complementary base sequence, histones, nucleic acid polymerase,nucleic acid binding protein.

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Contd.Contd.

Enzyme - substrate analogue, inhibitor,

cofactor.

Lectin - polysaccharide, glycoprotein, cell

surface receptor, cell.

Hormone, vitamin- receptor, carrier

protein.

Antibody - antigen, virus, cell.

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Applications in bioanalysis:

Affinity tags

a) GST tag.

b) HIS tag.

Antibody Immobilization.

DNA binding proteins.

Screening of Active Compounds.

Purification of receptors.

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GST- tags:

Size of 220 amino acids.

High affinity for glutathione.

An expression vector.

GST-fusion protein.

◦Binding.

◦Washing.

◦Elution.

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HIS-Tags:

N- or C-terminus of the protein.

Immobilized metal affinity

chromatography(IMAC).

Transition metal ion(Cu2+, Ni2+, Zn2+,

Co2+).

Electron donar group.

Matrix-chelating agents.

21Fig 5.

Contd.

Elution

◦pH value.

◦High salt concentration.

◦High displacement agent (e.g.,

imidazole)

◦Stronger metal-chelating agent (e.g.,

EDTA)

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Protein purification process.Fig No. 4

Antibody immobilisation:

Protein A and Protein G.

Antibody binding domains.

Fc region of polyclonal and monoclonal

IgG-type antibodies.

Co-purify host IgG.

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IgG antibody.Fig No.5

DNA binding proteins:

Ability to bind DNA.

Fusion proteins.

Specific affinity for heparin.

E.g.◦ Initiation factors, ◦ Elongation factors, ◦ Restriction endonucleases, ◦ DNA ligase, ◦ DNA and RNA polymerases.

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Screening of Active Compounds:Semen Armeniacae Amarum.

◦Treats cough and asthma.

◦β2-Adrenoceptor- the main target of

drugs for cough and asthma.

◦β2-AR affinity chromatographic

stationary phase Amygdalin is

retained.

26Chinese Science Bulletin | March 2008 | vol. 53 | no. 6 |

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The chromatogram of the total extracts of Semen Armeniacae Amarum on β2-AR chromatographic column

Graph.No. 2.

Chinese Science Bulletin | March 2008 | vol. 53 | no. 6 |

Purification of receptors:

mAChRs were solubilized with digitonin.

Ligand – Dexetimide. Matrix - Carboxy N-

hydroxysuccinimide esters linked agarose beads.

Elution – Atropine.

Radioiodinated mAChRs (mAChRp) were

revealed by autoradiography.

28The EMBO Journal Vol.2 No.4: © IRL Press Limited, Oxford, England.

Recents advances:

Synthetic de novo designed ligands.◦The rational method. ◦The combinatorial method.◦The combined method.

Biomimetic ligands.

Immunospecific affinity.

Two-dimensional electrophoresis (2D-

PAGE) and mass spectrometry (MS).

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Conclusion:

A modest chance of large-scale purification of proteinaceous pharmaceuticals.

Fewer purification steps.Increased product recovery. Identification and analysis of drug

receptors. Useful in drug development.

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References:

• Affinity Chromatography-Principles and

Methods by Amersham Biosciences.

• Affinity Chromatography by Jena Biosciences.

• Chinese Science Bulletin-March 2008, vol. 53;

no. 6.

• The EMBO Journal 1982, Vol.2; No.4: © IRL

Press Limited, Oxford, England.• Google image search.

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