Protein Purification

PEF utilises the capabilities of the ÄKTA Explorer and ÄKTAxpress chromatography systems for routine purification of recombinant proteins. Services available from PEF include:

Development of an effective protein purification and characterisation strategy

Access to a suite of automated chromatography systems, including:

  • ÄKTAprimeTM
  • ÄKTApurifierTM
  • ÄKTAexplorerTM
  • ÄKTAxpressTM
  • ÄKTApilotTM
  • Agilent 1200 Series HPLC

A range of chromatography columns for the following purification techniques:

  • Affinity Chromatography
  • Ion Exchange Chromatography
  • Size Exclusion Chromatography
  • Hydrophobic Interaction Chromatography
  • Reversed Phase Chromatography

An array of fusion tags available in each system:

  • Polyhistidine (6xHis, 10xHis, 12xHis)
  • Maltose Binding Protein (MBP)
  • Glutathione S-transferase (GST)
  • Thioredoxin (Trx)
  • Small Ubiquitin-like Modifier (SUMO)
  • Fluorescent proteins (Green, Cyan, Blue, Cherry, Citrine)

A variety of proteases for fusion tag removal:

  • Human Rhinovirus (HRV 3C)
  • Tobacco Etch Virus (TEVp)
  • SUMOp
  • Thrombin

For protein purification services, please contact us for a quote.

To view our list of available proteases and enzymes, please visit our UQ Resource Centre.

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:

  • Protein expression
  • Protein release and phase separation
  • Sample conditioning for purification
  • Chromatography
  • Characterisation
  • Formulation and storage


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

A key challenge in recombinant protein production is to maintain and store the target protein in a soluble and stable form. Protein aggregation can compromise protein function and thus it is necessary to overcome this challenge to generate functionally active protein.

Aggregates can be categorised as either “insoluble” (able to be removed by centrifugation or filtration) or “soluble” (not easily separated from native protein). Techniques such as analytical size exclusion chromatography (SEC), dynamic light scattering (DLS) and ultracentrifugation play an important role in identifying soluble aggregates.

Aggregation can occur at any stage of the production platform:

  • Protein Expression (e.g. inclusion body formation)
  • Protein Purification
    • Cell lysis and extraction
    • Chromatography
    • Buffer Exchange
    • Concentration
    • Storage

A number of strategies can be employed to overcome aggregation and promote protein stability.

The use of fusion tags, such as maltose binding protein (MBP) or thioredoxin (Trx), can impart solubility on proteins expressed heterologously in E. coliModifying expression culture conditions (e.g. reducing temperature) may also improve solubility by promoting correct folding.

Buffer conditions can be optimised to improve the target protein’s solubility during purification. Additives such as reducing agents (e.g. ß-mercaptoethanol, DTT), chaotropes (e.g. urea, guanidium-HCl), kosmotropes (e.g. ammonium sulphate, glycerol), detergents (e.g. tween, CHAPS), amino acids (arginine, glutamine) and ligands or cofactors (protein-dependent) can be used in low concentrations to stabilise the target protein’s native structure. Additionally, buffer pH and ionic strength also influence protein stability. Therefore it is often necessary to screen an array of buffer conditions and additives to determine the optimal buffering environment for the target protein. Once these stabilising conditions are known, they can be implemented throughout the purification process.

High protein concentration can compromise protein stability. Consequently, it may be necessary to maintain a low protein concentration by increasing the sample volume during lysis and chromatography. In situations where a high final protein concentration is required, stabilising buffer components can be included to avoid protein aggregation and maintain solubility.

Many proteins are unstable at 4˚C for more than a few days, so the preferred strategy is to store purified protein at -80˚C. However, subjecting proteins to repeated freeze-thaw cycles often leads to protein precipitation, so it is good practice to scout stability in advance. Buffer conditions that favour protein solubility at 4˚C may not necessarily prevent aggregation during freeze-thaw. Glycerol is often added to the protein sample as a cryoprotectant.

Other practices that reduce the propensity for proteins to aggregate include:

  • Performing all purification steps at 4˚C
  • Minimising sample handling
  • Avoiding time delays between purification steps
  • Reducing exposure to air-liquid interfaces (e.g. by avoiding bubble formation)

The fusion of a small protein or peptide (tag) to the protein of interest is a commonly used method to aid purification of recombinant proteins.

Fusion tags can improve protein expression, stability, resistance to proteolytic degradation and solubility. A wide range of fusion tags are available from small peptides to relatively large proteins, each with its own unique characteristics. Many solubility tags are engineered for use in bacterial expression systems to overcome poor protein solubility.

Fusion Tag Function Size (kDa) Description
Polyhistidine (e.g. 6xHis, 10xHis) Affinity 1-2 The most commonly used affinity tag, binds to metal ions
Strep-tag II Affinity 1 High affinity for engineered streptavidin
Thioredoxin (Trx) Solubility 12 Aids in refolding proteins that require a reducing environment
Small Ubiquitin-like Modifier (SUMO) Solubility 12 Contains a native cleavage sequence enabling tag removal with SUMO protease
Glutathione S-transferase (GST) Solubility, affinity 26 High affinity for glutathione, often needs to be removed due to large size
Maltose Binding Protein (MBP) Solubility, affinity 41 Binds to maltose, often needs to be removed due to large size

Combinatorial Fusion Tags

A combination of fusion tags can be used to maximise their functionality. A popular method is to utilise a solubility tag (e.g. GST or MBP) and an affinity tag (e.g. polyhistidine). This combination promotes soluble protein production and provides multiple options for affinity purification.

Fluorescent Proteins

Bioluminescent proteins are used in a wide range of biological applications from ultra-sensitive assay detection to in vivo imaging of cellular processes. The current range of bioluminescent proteins are variations of the wild-type Green Fluorescent Protein (GFP) derived from the jellyfish Aequorea victoria. Genetic variants featuring fluorescence emission spectral profiles spanning the blue, cyan, red, and yellow regions of the visible spectrum were developed by engineering specific mutations into GFP. These probes are used in a wide range of applications such as:

  • Fluorescent Resonance Energy Transfer (FRET)
  • Fluorescent Activated Cell Sorting (FACS)
  • Photoactivated Localisation Microscopy (PALM)
  • Fluorescence Recovery After Photobleaching (FRAP)
  • Confocal Microscopy

Tag removal

In many cases it is desirable to remove fusion tags during purification to restore native protein structure. Removal of the tag is achieved by including a cleavage site between the fusion tag and the gene sequence. Commonly used cleavage proteases include:

Protease Type Recognition Site
Human Rhinovirus 3C (HRV3C) Cysteine LEVLFQ/GP
Tobacco Etch Virus Protease (TEVp) Cysteine ENLYFQ/G
Ubiquitin-like Specific Protease (SUMOp) Cysteine Recognises tertiary structure
Thrombin Serine LVPA/GS

A number of challenges can be encountered during tag removal, including:

  • Incomplete cleavage
  • Difficulty in separating the protease and tag from the native protein
  • Loss of protein from the cleavage process
  • Loss of solubility following cleavage

Purification reviews

Recombinant protein expression and purification: a comprehensive review of affinity tags and microbial applications
Young CL, et alBiotechnology journal. 7(5), 620-634 (2012)

Immobilized-metal affinity chromatography (IMAC): a review
Block H, Maertens B, Spriestersbach A et alMethods in enzymology. 463, 439-473 (2009)

Current and prospective applications of metal ion-protein binding
Ueda EK, et al. Journal of chromatography. A. 988(1), 1-23 (2003)

Affinity Tags

An overview of enzymatic reagents for the removal of affinity tags
Waugh DS. Protein expression and purification. 80(2), 283-293 (2011)

Current strategies for the use of affinity tags and tag removal for the purification of recombinant proteins
Arnau J, et al. Protein expression and purification. 48(1), 1-13 (2006)

Making the most of affinity tags
Waugh DS. Trends in Biotechnology. 23(6), 316-320 (2005)

PEF offers upskilling for staff and students in all aspects of recombinant protein production. If you would like to enquire about training in any of the services offered by PEF please contact us.

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