The aim of a purification procedure is to obtain a highly pure and stable protein at an appropriate concentration in a buffer compatible with the intended application. Equal consideration must be given to the techniques used for purification in the early design phase of the project as well as during purification itself.  The methods and resulting yield are protein dependant and many contributing factors can affect the design, including:

  • Choice of expression system
  • Cloning strategy
  • Culture media
  • Protein solubility and stability
  • End use

The protein production process can be broken down into several steps:

Chromatography

Chromatography is the most powerful and commonly used means of purifying recombinant proteins. Each technique separates proteins based on different properties, so it is often advantageous to combine several types to maximise separation of the recombinant protein from host cell proteins.

Affinity and ion exchange chromatography are capable of handling large sample volumes and removing the bulk of contaminants, and thus are suitable for primary (capture) or intermediate purification steps. Size exclusion (gel filtration) can only handle small sample volumes and is best utilised as a final (polishing) step. Selecting the the most suitable techniques is important for a successful purification procedure, and depends upon the protein's unique characteristics. Commonly used techniques include:

Technique Stage Description
Affinity Chromatography (AC) Capture or Intermediate Based on a reversible interaction between the protein/affinity tag and a specific ligand
Ion Exchange Chromatography (IEX) Capture or Intermediate Separates proteins based on their net surface charge
Hydrophobic Interaction Chromatography (HIC) Intermediate Binding under high salt conditions, generally performed following an ammonium sulphate precipitation step
Size Exclusion Chromatography (SEC) Polishing Separates proteins based on their hydrodynamic volume (size)
Reverse Phase Chromatography (RPC) - High-resolution chromatography based on weak hydrophobic interactions. Harsh conditions generally only suitable for purification of peptides