Affinity Chromatography

Yongting Wang

Jan07

What is AC?

• Affinity chromatography (AC) is a technique enabling purification of a biomolecule with respect to biological function or individual chemical structure.

• AC is designed to purify a particular molecule from a mixed sample.

Matrix

Affinity Ligand

The resin

Examples of tags and ligands

• His-tag• FLAGTM peptide• Strep-tag• GST tag• Maltose binding protein fusion• Calmodulin binding protein fusion

• Transition metal ion• Monoclonal antibody• Biotin• Glutathione• Amylose• Ca2+

There are situations where you don’t need a tag.

Step 1. Loading affinity column.

Step 2. Proteins sieve through matrix of affinity beads.

Step 3. Proteins interact with affinity ligand with some binding loosely and others tightly.

Step 4. Wash off proteins that do not bind.

Step 5. Wash off proteins that bind loosely.

Step 6. Elute proteins that bind tightly to ligand and collect purified protein of interest.

http://www.bio.davidson.edu/Courses/genomics/method/Affinity.html

Affinity chromatography applied to recombinant proteins

Purity test

SDS-PAGE

Mass spectrometryN-terminal sequencing, etc.

Downstream of protein purification

• Biophysical characterization

• Biochemical analysis of activities

• Physiological relevance

• Pathological mechanisms

• etc.

Native

Unfolded

Fluorescence spectroscopy

Trp68

Trp42

Trp156

Trp130

N-terminal domain C-terminal domain

Diagrams of crystal structure of HgD

Example: Human lens crystallins

Human gene cloned into pET vector and expressed in E. coli.

Protein folding mechanisms

Electron microscopy IR spectroscopy

• In summary, Affinity chromatography is a method that will allow you to purify a particular molecule from a mixed sample so that further investigation of this molecule could be carried out.

• Thank you for your attention.

Three groups of properties of the target molecule are used in affinity chromatography:

1. Specific binding properties based on biological activity like:- Enzyme active sites- Receptor binding sites- Antibody binding sites etc.These are used together with the natural ligand or an analogue of it. Sometimes the analogue has a broader specificity and can be used for group separations.

2. Naturally occurring prosthetic groups like: polysaccharides etc.Such properties normally allow group separations only.

3. Molecules equipped with an affinity tag like:- Glutathione-S-Transferase (GST) - Oligo histidine etc. This group of properties is used almost exclusively for recombinant fusion proteins.