Protein Purification

• Before any particular protein can be

sequenced and characterized, its is necessary
to separate it from all of the other proteins in
the cell.
• This “purification” process involves
separating proteins based on their ionic
properties, their sizes, their hydrophobicity,
and their affinities for certain molecules
(ligands). Each successive step is referred to
as fractionation.
• Typically some form of column
chromatography is employed, in which the
solid phase (stationary phase) contains
molecules that in some way exploit the
differences among various proteins.
Ion-Exchange Chromatography
• This procedure is very similar to the
procedure that we talked about for
separating amino acids based on charge.
• The picture at the left represents ion-
exchange chromatography on a cation
exchanger. Notice that the bead is
negatively charged, and therefore the rate
of mobility of proteins loaded onto the
resin is proportional to the degree of
negative charge that they bear.
Size Exclusion Chromatography
• In size-exclusion chromatography (gel-filtration
chromatography), proteins migrate as a function of their
molecular weights.
• The solid matrix (beads) contains pores of various sizes.
The probability of entering the pores of the matrix is inversely
proportional to the size of the protein. In fact proteins that are
larger than a given size (depending on the resin that is used)
are totally excluded from entering the beads. Therefore, larger
proteins have a more direct route to the bottom of the column,
by simply going around all of the beads rather than entering
the beads.
• Notice in the figure at left that the large molecules elute
first.
• Size-exclusion chromatography can be used to determine the
molecular weight of a particular protein if appropriate
standards are available. As we’ll see in a couple of slides, the
elution volume is inversely proportional to the log of the
molecular weight.
Affinity Chromatrography
• In affinity chromatography, proteins are separated according
to their ability to bind to a specific ligand that is connected to
the beads of the resin.
• After the proteins that do not bind the ligand are washed
through the column, the bound protein of interest is eluted by a
solution containing free ligand.
 Salting Out
• The first step in purifying a protein is establishing a “crude extract.” This
requires that the membrane of the cell be ruptured by some technique.
• Upon rupturing the membrane and releasing the contents of the cell, the
insoluble debris is removed by centrifugation.
• Typically, one of the initial steps involves purifying proteins based on their
solubilties in varying concentrations of ammonium sulfate. The solubility of a
protein is sensitive to the concentrations of dissolved salts. The solubility of a
protein at low ionic strength generally increases with the salt concentration
(salting in). At high ionic strength, the solubilities of proteins decreases
(salting out).
• Many unwanted proteins can be eliminated by adjusting the salt concentration
in a solution containing the crude extract to just below the precipitation point
of the protein to be purified. In this case the protein to be purified remains in
solution, while many others are precipitated. Likewise, unwanted proteins can
be eliminated by adjusting the salt concentration to just over the precipitation
point of the protein of interest. In this case, the protein of interest will be
precipitated while many others will remain in solution.
 Specific Activity
• In order to purify a protein or any other substance, there must be a means of quantitatively detecting its
presence.
• Therefore, an assay must be determined. Typically assays are based on the function of a protein. For example,
a substrate like shown below might be used for an enzyme that cleaves an ester bond (esterase). One of the
products of this reaction has an intense absorption at 412 nm, which would allow for the detection of the
enzyme.
• You would be interested in how fast the enzyme cleaves this molecule (rate of color development), and how
much protein is being added to the reaction mixture. Your specific activity would be the rate divided by the
total amount of protein. The higher the specific activity the more pure your protein.
NO2

O
Intense absorption at 412 nm
(yellow)

O
NO2
Esterase
+
O

O
Hypothetical Purification
Electrophoresis
Electrophoresis is based on the migration of proteins in a charged field.
The force moving the macromolecules is the electrical potential, E.
µ = V/E, where µ is the electrophoretic mobility, and V is the velocity of the
particle.
µ = Z/f, where Z is the net charge of the molecule, and f is the frictional
coefficient. f is related to the the shape of the molecule, as well as its size.
Typically, the cross-linked polymer, polyacrylamide acts as the solid support.
Normally, proteins would be separated in proportion to their charge-to mass
ratio. The problem is that some proteins would migrate towards the anode,
while others would migrate toward the cathode; and, the migration would not
reflect size, but charge to mass.
The trick is to carry out the electrophoresis in the presence of sodium docecyl
sulfate, which is a detergent.
SDS binds to every protein in roughly the same proportion, which is about one
molecule for every two amino acid residues. SDS carries with it a negative
charge, and the cumulative negative charge renders the intrinsic net charge of
the protein insiginificant.
Therefore, every protein will have the same charge to mass ratio, which will
cause all proteins to migrate towards the cathode with a rathe that is dependent
on their sizes.
In contrast to gel-filtration, smaller molecules migrate faster than larger
molecules
Typically, this technique is not used to purify proteins, because SDS normally
denatures proteins. It is used to analyze the purity of proteins.
 Molecular Weight Determination

Shown above (on the left) is an SDS-polyacrylamide gel that has been stained with a dye in order to view the
proteins. Lane one of the left figure represents a set of standards, while lane 2 represents a protein that has
been purified. The molecular weight Mr of the protein can be estimated, by comparing its relative migration to
that of the standards. The relation is that the relative migration is dependent on the log of the molecular
weight.
 Isoelectric Focusing

Isoelectric focusing is a procedure that will allow the pI of a particular protein to be determined, and that will
separate proteins based on their respective pIs. In this technique, a mixture of proteins is applied to a gel that
contains a pH gradient. At the point along the gradient wherein the protein is no longer charged, it will cease to
migrate. This point is the pI of the protein, or the isoelectric point.
2D-Electrophoresis
2D-electrophoresis allows separation of proteins by both size
and isoelectric point. Each spot represents a different protein.
The horizontal represents the isoelectric focusing direction,
while the veritcal represents the SDS PAGE direction.