Protein purification is a series of processes intended to isolate one or a
few proteins from a complex mixture, usually cells, tissues or whole
organisms. Protein purification is vital for the characterization of the
function, structure and interactions of the protein of interest. The
purification process may separate the protein and non-protein parts of the
mixture, and finally separate the desired protein from all other proteins.
Separation of one protein from all others is typically the most laborious
aspect of protein purification. Separation steps usually exploit
differences in protein size, physico-chemical properties, binding
affinity and biological activity. The pure result may be termed protein
isolate. The methods used in protein purification
can roughly be divided into analytical and preparative methods. The distinction
is not exact, but the deciding factor is the amount of protein that can
practically be purified with that method. Analytical methods aim to detect
and identify a protein in a mixture, whereas preparative methods aim to
produce large quantities of the protein for other purposes, such as structural
biology or industrial use. In general, the preparative methods can be used in
analytical applications, but not the other way around.
Purpose Protein purification is either
preparative or analytical. Preparative purifications aim to produce a
relatively large quantity of purified proteins for subsequent use. Examples
include the preparation of commercial products such as enzymes, nutritional
proteins, and certain biopharmaceuticals. Analytical
purification produces a relatively small amount of a protein for a variety of
research or analytical purposes, including identification,
quantification, and studies of the protein's structure, post-translational
modifications and function. Pepsin and urease were the first proteins purified
to the point that they could be crystallized.
Preliminary steps = Extraction =
Depending on the source, the protein has to be brought into solution by breaking
the tissue or cells containing it. There are several methods to achieve this:
Repeated freezing and thawing, sonication, homogenization by high
pressure, filtration, or permeabilization by organic solvents.
The method of choice depends on how fragile the protein is and how sturdy
the cells are. Usually for most of the conventional purposes, column
chromatography is used to achieve purification. After this extraction
process soluble proteins will be in the solvent, and can be separated from cell
membranes, DNA etc. by centrifugation. The extraction process also extracts
proteases, which will start digesting the proteins in the solution. If the
protein is sensitive to proteolysis, it is usually desirable to proceed quickly,
and keep the extract cooled, to slow down proteolysis.
= Precipitation and differential solubilization =
In bulk protein purification, a common first step to isolate proteins is
precipitation with ammonium sulphate2SO4. This is performed by
adding increasing amounts of ammonium sulphate and collecting the different
fractions of precipitate protein. Ammonium sulphate can be removed by
dialysis.The hydrophobic groups on the proteins gets exposed to the atmosphere
and it attracts other protein hydrophobic groups and gets aggregated.
Protein precipitated will be large enough to be visible. One advantage of
this method is that it can be performed inexpensively with very large volumes.
The first proteins to be purified are water-soluble proteins. Purification of
integral membrane proteins requires disruption of the cell membrane in order
to isolate any one particular protein from others that are in the same
membrane compartment. Sometimes a particular membrane fraction can be
isolated first, such as isolating mitochondria from cells before purifying
a protein located in a mitochondrial membrane. A detergent such as sodium
dodecyl sulfate can be used to dissolve cell membranes and keep membrane
proteins in solution during purification; however, because SDS
causes denaturation, milder detergents such as Triton X-100 or CHAPS can be
used to retain the protein's native conformation during complete
purification. = Ultracentrifugation =
Centrifugation is a process that uses centrifugal force to separate mixtures
of particles of varying masses or densities suspended in a liquid. When a
vessel containing a mixture of proteins or other particulate matter, such as
bacterial cells, is rotated at high speeds, the inertia of each particle
yields an outward force proportional to its mass. The tendency of a given
particle to move through the liquid because of this force is offset by the
resistance the liquid exerts on the particle. The net effect of "spinning"
the sample in a centrifuge is that massive, small, and dense particles move
outward faster than less massive particles or particles with more "drag"
in the liquid. When suspensions of particles are "spun" in a centrifuge, a
"pellet" may form at the bottom of the vessel that is enriched for the most
massive particles with low drag in the liquid.
Non-compacted particles remain mostly in the liquid called "supernatant" and can
be removed from the vessel thereby separating the supernatant from the
pellet. The rate of centrifugation is determined by the angular acceleration
applied to the sample, typically measured in comparison to the g. If
samples are centrifuged long enough, the particles in the vessel will reach
equilibrium wherein the particles accumulate specifically at a point in
the vessel where their buoyant density is balanced with centrifugal force. Such
an "equilibrium" centrifugation can allow extensive purification of a given
particle. Sucrose gradient centrifugation — a
linear concentration gradient of sugar is generated in a tube such that the
highest concentration is on the bottom and lowest on top. Percoll is a
trademark owned by GE Healthcare companies. A protein sample is then
layered on top of the gradient and spun at high speeds in an ultracentrifuge.
This causes heavy macromolecules to migrate towards the bottom of the tube
faster than lighter material. During centrifugation in the absence of
sucrose, as particles move farther and farther from the center of rotation,
they experience more and more centrifugal force. The problem with this
is that the useful separation range of within the vessel is restricted to a
small observable window. Spinning a sample twice as long doesn't mean the
particle of interest will go twice as far, in fact, it will go significantly
further. However, when the proteins are moving through a sucrose gradient, they
encounter liquid of increasing density and viscosity. A properly designed
sucrose gradient will counteract the increasing centrifugal force so the
particles move in close proportion to the time they have been in the
centrifugal field. Samples separated by these gradients are referred to as "rate
zonal" centrifugations. After separating the protein/particles, the gradient is
then fractionated and collected. Purification strategies
Choice of a starting material is key to the design of a purification process. In
a plant or animal, a particular protein usually isn't distributed homogeneously
throughout the body; different organs or tissues have higher or lower
concentrations of the protein. Use of only the tissues or organs with the
highest concentration decreases the volumes needed to produce a given amount
of purified protein. If the protein is present in low abundance, or if it has a
high value, scientists may use recombinant DNA technology to develop
cells that will produce large quantities of the desired protein. Recombinant
expression allows the protein to be tagged, e.g. by a His-tag, to facilitate
purification, which means that the purification can be done in fewer steps.
In addition, recombinant expression usually starts with a higher fraction of
the desired protein than is present in a natural source.
An analytical purification generally utilizes three properties to separate
proteins. First, proteins may be purified according to their isoelectric
points by running them through a pH graded gel or an ion exchange column.
Second, proteins can be separated according to their size or molecular
weight via size exclusion chromatography or by SDS-PAGE analysis. Proteins are
often purified by using 2D-PAGE and are then analysed by peptide mass
fingerprinting to establish the protein identity. This is very useful for
scientific purposes and the detection limits for protein are nowadays very low
and nanogram amounts of protein are sufficient for their analysis. Thirdly,
proteins may be separated by polarity/hydrophobicity via high
performance liquid chromatography or reversed-phase chromatography.
Usually a protein purification protocol contains one or more chromatographic
steps. The basic procedure in chromatography is to flow the solution
containing the protein through a column packed with various materials. Different
proteins interact differently with the column material, and can thus be
separated by the time required to pass the column, or the conditions required
to elute the protein from the column. Usually proteins are detected as they
are coming off the column by their absorbance at 280 nm. Many different
chromatographic methods exist: = Size exclusion chromatography =
Chromatography can be used to separate protein in solution or denaturing
conditions by using porous gels. This technique is known as size exclusion
chromatography. The principle is that smaller molecules have to traverse a
larger volume in a porous matrix. Consequentially, proteins of a certain
range in size will require a variable volume of eluent before being collected
at the other end of the column of gel. In the context of protein purification,
the eluent is usually pooled in different test tubes. All test tubes
containing no measurable trace of the protein to purify are discarded. The
remaining solution is thus made of the protein to purify and any other
similarly-sized proteins. = Separation based on charge or
hydrophobicity = Hydrophobic interaction chromatography
HIC media is amphiphilic, with both hydrophobic and hydrophilic regions,
allowing for separation of proteins based on their surface hydrophobicity.
In pure water, the interactions between the resin and the hydrophobic regions of
protein would be very weak, but this interaction is enhanced by applying a
protein sample to HIC resin in high ionic strength buffer. The ionic
strength of the buffer is then reduced to elute proteins in order of decreasing
hydrophobicity. Ion exchange chromatography
Ion exchange chromatography separates compounds according to the nature and
degree of their ionic charge. The column to be used is selected according to its
type and strength of charge. Anion exchange resins have a positive charge
and are used to retain and separate negatively charged compounds, while
cation exchange resins have a negative charge and are used to separate
positively charged molecules. Before the separation begins a buffer is
pumped through the column to equilibrate the opposing charged ions. Upon
injection of the sample, solute molecules will exchange with the buffer
ions as each competes for the binding sites on the resin. The length of
retention for each solute depends upon the strength of its charge. The most
weakly charged compounds will elute first, followed by those with
successively stronger charges. Because of the nature of the separating
mechanism, pH, buffer type, buffer concentration, and temperature all play
important roles in controlling the separation.
Ion exchange chromatography is a very powerful tool for use in protein
purification and is frequently used in both analytical and preparative
separations. Free-flow-electrophoresis
Free-flow electrophoresis is a carrier-free electrophoresis technique
that allows preparative protein separation in a laminar buffer stream by
using an orthogonal electric field. By making use of a pH-gradient, that can
for example be induced by ampholytes, this technique allows to separate
protein isoforms up to a resolution of