Protein purification

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PROTEINS & PEPTIDES MUST BE
PURIFIED PRIOR TO ANALYSIS
Highly purified protein is essential for determination of
its amino acid sequence. Cells contain thousands of different
proteins, each in widely varying amounts. The
isolation of a specific protein in quantities sufficient for
analysis thus presents a formidable challenge that may
require multiple successive purification techniques.
Classic approaches exploit differences in relative solubility
of individual proteins as a function of pH (isoelectric
precipitation), polarity (precipitation with
ethanol or acetone), or salt concentration (salting out
with ammonium sulfate). Chromatographic separations
partition molecules between two phases, one mobile
and the other stationary. For separation of amino acids
or sugars, the stationary phase, or matrix, may be a
sheet of filter paper (paper chromatography) or a thin
layer of cellulose, silica, or alumina (thin-layer chromatography;
TLC).

Column Chromatography
Column chromatography of proteins employs as the
stationary phase a column containing small spherical
beads of modified cellulose, acrylamide, or silica whose
surface typically has been coated with chemical functional
groups. These stationary phase matrices interact
with proteins based on their charge, hydrophobicity,
and ligand-binding properties. A protein mixture is applied
to the column and the liquid mobile phase is percolated
through it. Small portions of the mobile phase
or eluant are collected as they emerge (Figure 4–1).
Partition Chromatography
Column chromatographic separations depend on the
relative affinity of different proteins for a given stationary
phase and for the mobile phase. Association between
each protein and the matrix is weak and transient.
Proteins that interact more strongly with the
stationary phase are retained longer. The length of time
that a protein is associated with the stationary phase is a
function of the composition of both the stationary and
mobile phases. Optimal separation of the protein of interest
from other proteins thus can be achieved by careful
manipulation of the composition of the two phases.
Size Exclusion Chromatography
Size exclusion—or gel filtration—chromatography separates
proteins based on their Stokes radius, the diameter
of the sphere they occupy as they tumble in solution.
The Stokes radius is a function of molecular mass
and shape. A tumbling elongated protein occupies a
larger volume than a spherical protein of the same mass.
Size exclusion chromatography employs porous beads
(Figure 4–2). The pores are analogous to indentations
in a river bank. As objects move downstream, those that
enter an indentation are retarded until they drift back
into the main current. Similarly, proteins with Stokes
radii too large to enter the pores (excluded proteins) remain
in the flowing mobile phase and emerge before
proteins that can enter the pores (included proteins).

Absorption Chromatography
For absorption chromatography, the protein mixture is
applied to a column under conditions where the protein
of interest associates with the stationary phase so
tightly that its partition coefficient is essentially unity.
Nonadhering molecules are first eluted and discarded.
Proteins are then sequentially released by disrupting the
forces that stabilize the protein-stationary phase complex,
most often by using a gradient of increasing salt
concentration. The composition of the mobile phase is
altered gradually so that molecules are selectively released
in descending order of their affinity for the stationary
phase.

Ion Exchange Chromatography
In ion exchange chromatography, proteins interact with
the stationary phase by charge-charge interactions. Proteins
with a net positive charge at a given pH adhere to
beads with negatively charged functional groups such as
carboxylates or sulfates (cation exchangers). Similarly,
proteins with a net negative charge adhere to beads with
positively charged functional groups, typically tertiary or
quaternary amines (anion exchangers). Proteins, which
are polyanions, compete against monovalent ions for
binding to the support—thus the term “ion exchange.”
For example, proteins bind to diethylaminoethyl
(DEAE) cellulose by replacing the counter-ions (generally
Cl− or CH3COO−) that neutralize the protonated
amine. Bound proteins are selectively displaced by gradually
raising the concentration of monovalent ions in the mobile phase. Proteins elute in inverse order of the
strength of their interactions with the stationary phase.
Since the net charge on a protein is determined by
the pH , sequential elution of proteins
may be achieved by changing the pH of the mobile
phase. Alternatively, a protein can be subjected to consecutive
rounds of ion exchange chromatography, each
at a different pH, such that proteins that co-elute at one
pH elute at different salt concentrations at another pH.

Hydrophobic Interaction Chromatography
Hydrophobic interaction chromatography separates
proteins based on their tendency to associate with a stationary
phase matrix coated with hydrophobic groups
(eg, phenyl Sepharose, octyl Sepharose). Proteins with
exposed hydrophobic surfaces adhere to the matrix via
hydrophobic interactions that are enhanced by a mobile
phase of high ionic strength. Nonadherent proteins are
first washed away. The polarity of the mobile phase is
then decreased by gradually lowering the salt concentration.
If the interaction between protein and stationary
phase is particularly strong, ethanol or glycerol may be
added to the mobile phase to decrease its polarity and
further weaken hydrophobic interactions.
Affinity Chromatography
Affinity chromatography exploits the high selectivity of
most proteins for their ligands. Enzymes may be purified by affinity chromatography using immobilized substrates,
products, coenzymes, or inhibitors. In theory,
only proteins that interact with the immobilized ligand
adhere. Bound proteins are then eluted either by competition
with soluble ligand or, less selectively, by disrupting
protein-ligand interactions using urea, guanidine
hydrochloride, mildly acidic pH, or high salt concentrations.
Stationary phase matrices available commercially
contain ligands such as NAD+ or ATP analogs. Among
the most powerful and widely applicable affinity matrices
are those used for the purification of suitably modified
recombinant proteins. These include a Ni2+ matrix
that binds proteins with an attached polyhistidine “tag”
and a glutathione matrix that binds a recombinant protein
linked to glutathione S-transferase.
Peptides Are Purified by Reversed-Phase
High-Pressure Chromatography
The stationary phase matrices used in classic column
chromatography are spongy materials whose compressibility
limits flow of the mobile phase. High-pressure liquid
chromatography (HPLC) employs incompressible
silica or alumina microbeads as the stationary phase and
pressures of up to a few thousand psi. Incompressible
matrices permit both high flow rates and enhanced resolution.
HPLC can resolve complex mixtures of lipids or
peptides whose properties differ only slightly. Reversedphase
HPLC exploits a hydrophobic stationary phase of aliphatic polymers 3–18 carbon atoms in length. Peptide
mixtures are eluted using a gradient of a water-miscible
organic solvent such as acetonitrile or methanol.

Protein Purity Is Assessed by
Polyacrylamide Gel Electrophoresis
(PAGE)
The most widely used method for determining the purity
of a protein is SDS-PAGE—polyacrylamide gel
electrophoresis (PAGE) in the presence of the anionic
detergent sodium dodecyl sulfate (SDS). Electrophoresis
separates charged biomolecules based on the rates at
which they migrate in an applied electrical field. For
SDS-PAGE, acrylamide is polymerized and crosslinked
to form a porous matrix. SDS denatures and
binds to proteins at a ratio of one molecule of SDS per
two peptide bonds. When used in conjunction with 2-
mercaptoethanol or dithiothreitol to reduce and break
disulfide bonds (Figure 4 –3), SDS separates the component
polypeptides of multimeric proteins. The large
number of anionic SDS molecules, each bearing a
charge of −1, on each polypeptide overwhelms the
charge contributions of the amino acid functional
groups. Since the charge-to-mass ratio of each SDSpolypeptide
complex is approximately equal, the physical
resistance each peptide encounters as it moves
through the acrylamide matrix determines the rate of
migration. Since large complexes encounter greater resistance,
polypeptides separate based on their relative
molecular mass (Mr). Individual polypeptides trapped
in the acrylamide gel are visualized by staining with
dyes such as Coomassie blue (Figure 4–4).
Isoelectric Focusing (IEF)
Ionic buffers called ampholytes and an applied electric
field are used to generate a pH gradient within a polyacrylamide
matrix. Applied proteins migrate until they
reach the region of the matrix where the pH matches
their isoelectric point (pI), the pH at which a peptide’s
net charge is zero. IEF is used in conjunction with SDSPAGE
for two-dimensional electrophoresis, which separates
polypeptides based on pI in one dimension and
based on Mr in the second . Two-dimensional
electrophoresis is particularly well suited for separating
the components of complex mixtures of proteins.

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