(ligand) is bound to an insoluble support. When a crude mixture of proteins contain-
ing the protein of interest is passed through the column, the ligand binds the protein
to the matrix whilst all other proteins pass through the column. The bound protein
can then be eluted from the column by changing the pH, increasing salt strength or
passing through a high concentration of unbound free ligand. For example, the
protein concanavalin A (con A) binds strongly to glucose. An affinity column using
glucose as the ligand can therefore be used to bind con A to the matrix, and the con
A can be recovered by passing a high concentration of glucose through the column.
Affinity chromatography is covered in detail in Section 11.8.Hydrophobicity
Proteins differ in the amount of hydrophobic amino acids that are present on their
surface. This difference can be exploited in salt fractionation (see above) but can also
be used in a higher resolution method using hydrophobic interaction chromatog-
raphy (HIC) (Section 11.4.3). A typical column material would be phenyl-Sepharose,
where phenyl groups are bonded to the insoluble support Sepharose. The protein
mixture is loaded on the column in high salt (to ensure hydrophobic patches are
exposed) where hydrophobic interaction will occur between the phenyl groups on the
resin and hydrophobic regions on the proteins. Proteins are then eluted by applying a
decreasing salt gradient to the column and should emerge from the column in order
of increasing hydrophobicity. However, some highly hydrophobic proteins may not
even be eluted in the total absence of salt. In this case it is necessary to add a small
amount of water-miscible organic solvent such as propanol or ethylene glycol to the
column buffer solution. This will compete with the proteins for binding to the
hydrophobic matrix and will elute any remaining proteins.8.3.5 Engineering proteins for purification
With the ability to clone and overexpress genes for proteins using genetic engineering
methodology has also come the ability to aid considerably the purification process by
manipulation of the gene of interest prior to expression. These manipulations are carried
out either to ensure secretion of the proteins from the cell or to aid protein purification.Ensuring secretion from the cell
For cloned genes that are being expressed in microbial or eukaryotic cells, there are a
number of advantages in manipulating the gene to ensure that the protein product is
secreted from the cell:- To facilitate purification: Clearly if the protein is secreted into the growth medium,
there will be far fewer contaminating proteins present than if the cells had to be
ruptured to release the protein, when all the other intracellular proteins would also
be present. - Prevention of intracellular degradation of the cloned protein: Many cloned proteins are
recognised as ‘foreign’ by the cell in which they are produced and are therefore
degraded by intracellular proteases. Secretion of the protein into the culture medium
should minimise this degradation.
326 Protein structure, purification, characterisation and function analysis