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Protein Expression and Purification Core FacilityProtein Purification

Purification

Tag
Binding matrix
elution compound (conc.)
His6-tag (sometimes up to His10)
Immobilised, chelated Ni2+ or Cu2+
Imidazole (250-500 mM) or lowering pH to ~pH 5
Glutathione S-transferase (GST)
Immobilized Glutathione (GSH)
Reduced GSH (10-20 mM)
Maltose-binding protein (MBP)
Amylose
Maltose (10 mM)
Strep-tag / StrepII-tag
Strep-TactinTM
Desthiobiotin (2.5 mM)
Cellulose-binding domain (CBD)
Cellulose
Ethylenglycol (50% v/v)
Calmodulin-binding peptide (CBP)
Immobilised Calmodulin
EGTA (2 mM)
Intein
Chitin
DTT (30-50 mM)

These tags are used under non-denaturing conditions, purification via His6-tag can also be performed in denaturing conditions, e.g. if the protein is mostly insoluble.

To increase purity, usually a second binding/elution process is utilized; most frequently an ion-exchange (IEX) chromatography step is the method of choice. Depending on the pI-values and differences between target and contaminant proteins, anion-exchange or cation-exchange matrices are used in low salt concentrations to bind the target protein while contaminant proteins ideally bind weakly. With increasing salt concentrations, the interactions of charged groups on the surface of the proteins become weaker and eventually all proteins elute at a characteristic salt concentration range and can be separated.

Salt-dependent binding of a protein can also be utilized in hydrophobic interaction chromatography (HIC), while in this case an inverse gradient from high salt to low salt concentration is applied. Here the binding is mediated by hydrophobic side chains and hydrophobic groups on the resin.

As a final polishing step, gel filtration chromatography can be used to increase the purity and homogeneity of the protein sample. The remaining proteins are separated on a porous matrix according to their size, thus not only contaminant protein with different size than the target protein but also different oligomeric forms of the same (apparently pure) protein can be separated. This can be critical for protein crystallisation experiments.

Note, that gel filtration is not only a preparative method but also provides valuable information for the analysis of protein-protein interactions as you can study the association properties of one or multiple proteins in different conditions.