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Education Centre
About the purification of biomolecules
Purpose of purification
Developing purification protocols
How to combine purification steps
Purification development - summary
LC techniques
Affinity Chromatography
Animation of affinity chromatography
Basic principles of affinity chromatography
The affinity chromatography separation mechanism
The affinity chromatography experiment
Types of target molecular properties
Types of affinity chromatography ligands
Suitable conditions for binding/elution
Affinity chromatography applied to recombinant proteins
Affinity chromatography in Practice
Affinity chromatography technique profile
What is affinity chromatography?
Desalting & Gel Filtration
Hydrophobic interaction chromatography
Ion exchange chromatography
Reversed phase chromatography
Protein Purifier software
BioProcess™ Glossary

Affinity chromatography applied to recombinant proteins

The purification of recombinant proteins may be drastically simplified by fusing the gene for an affinity tag (or handle) with the gene for the recombinant.
The host will then express the recombinant protein tagged with the affinity handle and affinity chromatography techniques can be applied to isolate and purify this so-called fusion protein (Fig 6.1.). Though not always necessary, the affinity tag can be removed by special cleave-off enzymes after the purification.

Fig 6.1. Production and purification of fusion proteins.


The merits of this fusion protein technique are:
  • The ligand/affinity tag system can in principle be applied to any recombinant protein.
  • The purification protocol is reduced to one step.
  • Conditions used become standardized.
  • Very little (if any) optimization work is needed.

Figures 6.2 and 6.3 below show the workflow for purification of GST-tagged and (His)6-tagged fusion proteins:

Fig 6.2. Schematic overview of GST fusion protein purification using GSTrap™.



Fig 6.3. Schematic overview of (His)6 fusion protein purification using HiTrap™ Chelating HP