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Protein purification methods
Concepts
Methods
Applications
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Recombinant protein purification: step by step
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.
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Chromatography columns in protein purification
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
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.
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Affinity chromatography: fusion tags
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 Stransferase
(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
Fusion tags can improve protein expression, stability, resistance to proteolytic
degradation and solubility.
• Fusion tag orientation (N- or C-terminus)
• Combinatorial fusion tags (Trx/GST/MBP with an affinity tag, e.g. 6xHis)
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Variety of proteases for fusion tag removal
• Human Rhinovirus (HRV 3C)
• PreScission protease
• Tobacco Etch Virus (TEVp)
• SUMOp
• Thrombin
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Column chromatography instrumentation
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Protein characterization: an aggregation problem
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.
Detection of protein aggregation
• Analytical size-exclusion chromatography (SEC)
• Dynamic light scattering (DLS)
• Analytical ultracentrifugation (AUC)
Troubleshooting
• Culture conditions (e.g. reducing temperature)
• Buffer composition (ionic strenght, pH, reducing agents)
• Presence co-factors (Acetyl-CoA, metal ions)
• Fusion tags (Trx, MBP, SUMO)
• Minimising sample handling
• Avoiding time delays between purification steps
• Performing purification steps at 4°C
• Store purified proteins in -80°C
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Protein quality control (QC) analyses
High purity and homogeneity of the protein sample are crucial for the
downstream processes to be successful.
• Dynamic light scattering (DLS): To characterize the polydispersity of sample
Identification of different oligomeric forms or
aggregates, which are preventing crystallization
• Differential scanning fluorimetry (DSF): analysis of protein stability
To characterize the stability of the protein in different
buffers and in the presence of different ligands, which
stabilize the protein for crystallization
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Success stories
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Development of expression and purification protocol
for Schistosoma mansoni HDAC8: mini-scale tests
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Large-scale production and crystallization of smHDAC8
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Conclusions
• The project design is ultimately determined by the
end-use of the recombinant protein
• The overall success of a project lies in an effective
project design
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Questions
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Dr. Martin Marek
Loschmidt Laboratories
Faculty of Science, MUNI
Kamenice 5, bld. A13, room 332
martin.marek@recetox.muni.cz