Affinity Chromatography

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Text of Affinity Chromatography

Affinity Chromatography A Bioanalytical ToolGuided by: Mr. S.A.Pishwikar Sir. Asst. Professor. Presentation By: Mr. Arpit H. Patel. M.Pharm 1st sem.

Department of Quality Assurance, Bharati Vidyapeeth College Of Pharmacy, Kolhapur.1

Outlines of the Seminar:y y y y y y y y

Abbrevations. Introduction. Principle. Steps of the technique. Applications in Bioanalysis. Recent advances. Conclusion. References.2

Abreviations:y y y y y y

GST Glutathione S- transferace. HIS Polyhistidine/hexahistidine. EDTA Ethylene diamine tetra acetic acid. IgG Immunoglobulin G. 2 AR- 2 Adrenoreceptors. mAChR Muscarinic acetycholine receptors.

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Introduction:Various Purification Techniques.

Gel filtration

Hydrophobic interaction

Ion exchange

Affinity

Various Biomolecule Purification Techniques. Fig No. 1.

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Affinity chromatography.

Unique purification technique. Separates active biomolecules.Defination.y y

Liquid chromatographic technique. biological interaction.

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Components of affinity chromatography.

Matrix

Spacer arm

Ligand

Molecule of interest

Design of affinity chormatography model. Fig No. 2

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Principle:y y y

Liquid adsorption chromatography. Reversible adsorption. Biospecific interaction. Electrostatic interactions. Van der Waals' forces. Hydrogen bonding.

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Ligand is immobilised to an matrix.7

Steps of Affinity Chromatography:y

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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y

Properties of Matrix.An inert support. Good flow properties. An open pore structure. Stability. Degree of crosslinking.E.g. Sepharose.

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y

Selection of Ligand :Binds reversibly. Chemically compatability towards matrix. Attachment site.

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y

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. 12

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. Buffer pH. Ionic strength. Polarity.

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o

o

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a) b) c)

Equilibration. Sample application and wash. Elution.

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y y y y

the level of complexity qualitative information quantitative information total column performance

Time

Graph No. 1. 16

Some typical biological interactions:y

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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y

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Contd.

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Enzyme - substrate analogue, inhibitor, cofactor.

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Lectin - polysaccharide, glycoprotein, cell surface receptor, cell.

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Hormone, vitamin- receptor, carrier protein. Antibody - antigen, virus, cell.

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Applications in bioanalysis:y

Affinity tagsa) GST tag. b) HIS tag.

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Antibody Immobilization. DNA binding proteins. Screening of Active Compounds. Purification of receptors.

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GST- tags:yy y y

Size of 220 amino acids.High affinity for glutathione. An expression vector. GST-fusion protein. Binding. Washing. Elution.20

HIS-Tags:yy

N- or C-terminus of the protein.Immobilized metal affinity chromatography(IMAC).

y y y

Transition metal ion(Cu2+, Ni2+, Zn2+, Co2+). Electron donar group. Matrix-chelating agents.

Fig 5.

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Contd.

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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

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Antibody immobilisation:y y y

Protein A and Protein G. Antibody binding domains. Fc region of polyclonal and monoclonal IgGtype antibodies.

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Co-purify host IgG.

IgG antibody. Fig No.5 24

DNA binding proteins:y

Ability to bind DNA.

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Fusion proteins.Specific affinity for heparin. E.g.Initiation factors, Elongation factors, Restriction endonucleases, DNA ligase, DNA and RNA polymerases.25

Screening of Active Compounds:y

Semen Armeniacae Amarum.Treats cough and asthma.2

-Adrenoceptor- the main target of drugs

for cough and asthma.2

-AR affinity chromatographic stationary Amygdalin is retained.

phase

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

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The chromatogram of the total extracts of Semen Armeniacae Amarum on 2-AR chromatographic column Graph.No. 2.

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

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Purification of receptors:y y

mAChRs were solubilized with digitonin. Ligand Dexetimide.

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Matrix - Carboxy N-hydroxysuccinimide esters linked agarose beads.Elution Atropine. Radioiodinated mAChRs (mAChRp) were revealed by autoradiography.

y y

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

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Recents advances:y

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

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Biomimetic ligands.

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Immunospecific affinity.Two-dimensional electrophoresis (2D-PAGE) and mass spectrometry (MS).29

Conclusion:y y y y y

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 andMethods 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: IRLPress Limited, Oxford, England.

Google image search.31