section 3.3
Separation of Proteins
Ion exchange (desorption)
Steps involved in the ion exchange chromatographic separation of proteins,
(a) At an appropriate pH, a protein with positively charged groups is
adsorbed to the resin by electrostatic bonding, (b) As the concentration of
NaCl is increased in the solvent flowing through the column, Na+
competes with the positively charged protein for binding with the
negatively charged groups or the resin, and desorption of the protein from
the resin particles occurs, (c) The released protein is carried away with the
flow of the solvent.
matrix (e.g., enzyme-substrate, hormone-receptor,
antigen-antibody interactions). The analogues on the col-
umn are usually small molecules resembling enzyme sub-
strates, hormones, or antigens. When a protein solution
is applied to the column, only those proteins with a
high affinity for the matrix are bound. Proteins that do
not specifically bind pass rapidly through the column.
The bound proteins can be eluted by altering the pH or
ionic strength of the eluent or by adding excess quanti-
ties of the ligand, e.g., hormone, antigen, or enzyme sub-
strate or inhibitor. Affinity chromatography is useful in
the purification of enzymes, hormones and their receptor
sites, immunoglobulins (Chapter 35), and nucleic acids
(Chapter 23).
Affinity Tag Chromatography
Affinity tag chromatography permits purification of re-
combinant proteins from growth media or from cell
lysates. New chromatography techniques take advantage
of DNA cloning that produces recombinant fusion pro-
teins and allows such proteins to be easily purified. Re-
combinant proteins can be engineered to contain affinity
tag sequences to create a fusion protein. The tag possesses
unique affinity characteristics that serve as the basis for
subsequent purification. Affinity chromatography is car-
ried out using the immobilized ligand of the tag, which
yields a highly purified fusion protein. A variety of affin-
ity tag sequences are used such as
for metal
chelate separation, enzyme tags that allow isolation using
immobilized substrate, or epitope sequences for separa-
tion by an immobilized monoclonal antibody. An enzyme
cleavage site is usually included between the tag and pro-
tein for removal of the tag from the fusion protein after
purification. Once an effective purification strategy has
been established for one fusion protein, it can be used for
any protein that is engineered to include the same tag.
One example of this method is the use of glutathione
S-transferase (GST) as the affinity tag. The fusion protein
is engineered to contain a thrombin cleavage site between
GST and the protein N terminus for subsequent removal
of the GST (Figure 3-4). The sample is first run through a
capture column consisting of the natural substrate for GST,
glutathione bound to agarose. The column is then washed,
this removing all unbound molecules and cellular debris.
A large amount of free glutathione is then added to the col-
umn. The free glutathione outcompetes the agarose-bound
glutathione for GST causing elution of the fusion protein.
The eluted protein is then run through a second column
consisting of immobilized thrombin, which will remove
the GST affinity tag by cutting at the thrombin cleavage
site. An enzyme column is used instead of added enzyme
because the bound enzyme maximizes the interaction be-
tween substrate and enzyme. Furthermore, it eliminates
the need for an enzyme removal step and enables the en-
zyme column to be reused. The eluant from the thrombin
column is passed through a gel filtration column that sep-
arates the protein from the affinity tag based on molecular
size. Finally, the eluant is fractionated and analyzed for
Separation by Electrophoresis
Electrophoresis separates charged proteins on the basis of
their different mobilities in an electric field. When a solu-
tion of proteins is subjected to an electrical potential, the
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