Affinity Chromatography Notes

Purification of Proteins by Affinity Chromatography

Principles of Chromatography

• Definition: Chromatography separates molecules based on their differential partitioning between two phases.

  • Stationary Phase: Solid and immobile, typically found in molecular biology.

  • Mobile Phase: Liquid phase that flows during the process.

Classical Column Chromatography

1. The mixture to be separated is dissolved in the mobile phase.

2. The mobile phase is continuously added.

3. Components separate in the stationary chromatography column, collected at the column's bottom.

Partitioning Coefficient (α)

• Definition: Fraction of molecules (solutes) adsorbed on the stationary phase.

• Example: If 90% of the solute is adsorbed, then α = 0.9.

• Mathematical Representation: $\alpha = \frac{[adsorbed \; solute]}{[total \; (adsorbed + desorbed) \; solute]}$

Types of Chromatography Used in Protein Separation

• Column chromatography (low pressure, HPLC)

• Ion exchange chromatography

• Inorganic adsorbents (bentonite, hydroxyapatite, titanium oxide)

• Hydrophobic chromatography

• Gel filtration (size exclusion chromatography)

• Affinity chromatography (various ligands, antibodies, etc.)

Stationary Phase Materials

• Cellulose

• Crosslinked agarose

• Synthetic polymers

• Inorganic materials (HAP, glass)

• Magnetic beads

• Plastic surfaces, membranes, array surfaces

• Micro-beads, macro-beads, nanoparticles

Mechanism of Chromatography

• For$\alpha = 0$: No adsorption.

• For$\alpha = 1$: All proteins are adsorbed.

• For$0 < \alpha < 1$: Proteins partially adsorb and move down the column.

• In affinity chromatography, $\alpha$ must be ≥ 0.8 for effective adsorption. —> in our case, much higher, around 20

Affinity Chromatography

• Definition: Separates proteins based on reversible interactions with specific ligands on the stationary phase.

• Selectivity: Can achieve several thousand-fold purification in a single step while recovering active proteins.

• Only chromatography that enables purification of proteins on basis of biological function or individual structure.

• Versatility: Suitable for isolating pure proteins from crude samples, even at low concentrations.

  • Can be specific for a single protein or a class of proteins, e.g., nucleic acid binding proteins.

Requirements for Affinity Chromatography Matrix

1. Ligand attachment must be covalent.

2. Ligand needs a spacer arm to avoid steric hindrance.

3. Minimal non-specific interactions.

Affinity Chromatography Examples

• Maltose and Maltose Binding Protein

• Glutathione and Glutathione S-transferase ← what we used

• Nickel (Ni²⁺) with Hexa His tag

• Avidin/streptavidin with Biotinylated Protein

• Antibody-antigen interactions

• Protein A/G/L with various antibodies (IgG, IgA, IgM)

Advantages of Affinity Chromatography

• Purification factor >1000x with high recovery.

• Mild binding and wash conditions preserve protein integrity.

• Elution options:

  1. Competition with free ligands (reduced $\alpha$ (close to 1 —> 0) leads to protein dissociation).

  2. Strong interactions may require harsher conditions such as increased salt or pH changes. (AB/antigen immunopurification)

Practical Steps in Affinity Chromatography

1. Select chromatography media (pre-activated, ready for ligand attachment).

2. Choose format: pre-packed columns or batch mode.

3. Optimize flow rate for effective binding and recovery.

4. Equilibrate media with binding buffer.

5. Apply sample and wash (sample volume does not impact separation).

6. Elution by changing ionic strength or pH, or competitive elution agents.

Affinity Chromatography Workflow

• Steps:

  1. Equilibrate

  2. Load sample

  3. Wash

  4. Elute

Batch Adsorption

• Definition: Simplistic method that does not use columns but has strict requirements.

• Requirement: α of the protein should be very close to 1 for effective binding. (V. strong interaction between protein and ligand). All other proteins must have very low.

GST Fusion Proteins Affinity Purification

• Use of glutathione immobilized on agarose or magnetic beads.

• Binding achieved under mild conditions.

• Elution performed using excess reduced glutathione.

Structure of Glutathione

• Structure includes:

  • A peptide binding site complementary to Glutathione S-transferase (GST).

Parameters Affecting Binding of GST-Fusion Proteins

• Binding kinetics between glutathione and GST is slow; use low flow rate or batch method for best results.

• Varying volumes/times for elution may enhance yield; higher glutathione concentrations (20-50mM) may be beneficial.