Lecture 8: Protein purification
– Protein purification
Column chromatography
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After the initial fractionation steps we move to column chromatography.
The mixture of substances (proteins) to be fractionated is dissolved in a liquid or
gaseous fluid called the mobile phase.
This solution is passed through a column consisting of a porous solid matrix called
the stationary phase. These are sometimes called resins when used in liquid
chromatography.
The stationary phase has certain physical and chemical characteristics that allow it to
interact in various ways with different proteins.
Common types of chromatographic stationary phases
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–
–
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Ion exchange
Hydrophobic
Gel filtration
Affinity
Ion exchange chromatography
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Ion exchange resins contain charged groups.
If these groups are acidic in nature they interact with positively charged proteins and
are called cation exchangers.
+
CH2-COO
+
+
CH -COO2
•
+
Positively
charged (basic)
protein or enzyme
CM cellulose
If these groups are basic in nature, they interact with negatively charged molecules
cation
exchanger
and
are called
anion exchangers.
CH2-CH2 -NH+(CH2CH2) CH2-CH2 -NH+(CH2CH2)
DEAE cellulose
anion exchanger
Negatively
charged (acidic)
protein or enzyme
Ion exchange chromatography
For protein binding, the pH is fixed (usually near neutral) under low salt
conditions. Example cation exchange column…
+
CH2-COO- +
+
CH2-COO-+
CM cellulose
cation exchanger
-
-
-
-
Positively charged protein
or enzyme bind to the
column
Negatively
charged proteins
pass through the
column
Ion exchange chromatography
To elute our protein of interest, add increasingly higher amount of salt
(increase the ionic strength). Na+ will interact with the cation resin and Clwill interact with our positively charged protein to elute off the column.
+
CH2-COO +
CH -COO- +
2
+
+ Increasing
[NaCl] of the
elution buffer
CM cellulose
cation exchanger
Na+ Na+2
CH2-COO
+
Na
CH -COO
2
CM cellulose
cation exchanger
Na+2
ClCl- +
+
Cl
+
Cl +
Ion exchange chromatography
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Proteins will bind to an ion exchanger with different affinities.
As the column is washed with buffer, those proteins relatively low affinities
for the ion exchange resin will move through the column faster than the
proteins that bind to the column.
The greater the binding affinity of a protein for the ion exchange column, the
more it will be slowed in eluting off the column.
Proteins can be eluted by changing the elution buffer to one with a higher
salt concentration and/or a different pH (stepwise elution or gradient
elution).
Cation exchangers bind to proteins with positive charges.
Anion exchangers bind to proteins with negative charges.
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Figure 6-6 Ion exchange chromatography
using stepwise elution.
Ion exchange chromatography
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Gradient elution can improve the washing of ion exchange columns.
The salt concentration and/or pH is continuously varied as the column is eluted so as
to release sequentially the proteins bound to the column.
The most widely used gradient is the linear gradient where the concentration of
eluant solution varies linearly with the volume of the solution passed.
The solute concentration, c, is expressed as
c = c2 - (c2 - c1)f
c1 = the initial concentration of the solution in the mixing chamber
c2 = the concentration of the reservoir chamber
f = the remaining fraction of the combined volumes of the solutions initially present in
both reservoirs.
Page 135
Figure 6-7 Device for generating a linear
concentration gradient.
c = c2 - (c2 - c1)f
Page 135
Figure 6-8 Molecular formulas of cellulosebased ion exchangers.
Table 6-2 Some Biochemically Useful Ion
Exchangers.
Ion exchange chromatography
• Ion exchangers can be cellulosic ion exchangers and
gel-type ion exchangers.
• Cellulosic ion exchangers most common.
• Gel-type ion exchangers can combine with gel filtration
properties and have higher capacity.
• Disadvantage-these materials are easily compressed so
eluant flow is low.
• There are other materials derived from silica or coated
glass beads that address this problem.
Gel filtration chromatography
• Also called size exclusion chromatography or
molecular sieve chromatography.
How does it work? If we assume proteins are spherical…
size
Molecular mass
(daltons)
10,000
30,000
100,000
Gel filtration chromatography
flow
Gel filtration chromatography
flow
Gel filtration chromatography
flow
Gel filtration chromatography
flow
Gel filtration chromatography
flow
Gel filtration chromatography
• The molecular mass of the smallest molecule unable to penetrate
the pores of the gel is at the exclusion limit.
• The exclusion limit is a function of molecular shape, since elongated
molecules are less likely to penetrate a gel pore than other shapes.
• Behavior of the molecule on the gel can be quantitatively
characterized.
Total bed volume of the column
Vt = Vx + V0
Vx = volume occupied by gel beads
V0 = volume of solvent space surrounding gel; Typically 35%
Gel filtration chromatography
• Elution volume (Ve) is the volume of a solvent required to elute a
given solute from the column after it has first contacted the gel.
• Relative elution volume (Ve/V0) is the behavior of a particular
solute on a given gel that is independent of the size of the column.
• This effectually means that molecules with molecular masses
ranging below the exclusion limit of a gel will elute from a gel in the
order of their molecular masses with the largest eluting first.
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Figure 6-9 Gel filtration chromatography.
Page 138
Figure 6-10
Molecular mass determination
by gel filtration chromatography.
Page 138
Table 6-3 Some Commonly Used Gel
Filtration Materials.
Gel filtration chromatography
• Elution volume (Ve) is the volume of a solvent required to elute a
given solute from the column after it has first contacted the gel.
• Relative elution volume (Ve/V0) is the behavior of a particular
solute on a given gel that is independent of the size of the column.
• This effectually means that molecules with molecular masses
ranging below the exclusion limit of a gel will elute from a gel in the
order of their molecular masses with the largest eluting first.
Affinity chromatography
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•
Many proteins can bind specific molecules very tightly but noncovalently.
We can use this to our advantage with affinity chromatography.
Glucose (small dark blue molecule) binding to hexokinase.
The enzyme acts like a jaw and clamps down on the
substrate (glucose)
Affinity chromatography
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How does it work?
Ligand - a molecule that specifically binds to the protein of interest.
Inert support
+
+
Spacer arms
Affinity material
prepared
Inert support
Ligand
Affinity chromatography
Inert support
Mixture of proteins
Inert support
Unwanted proteins
Affinity chromatography
Inert support
Elute with competitive ligand.
Inert support
Remove from competitive ligand
by dialysis.
Affinity chromatography
• To remove the protein of interest from the column, you
can elute with a solution of a compound with higher
affinity than the ligand (competitive)
• You can change the pH, ionic strength and/or
temperature so that the protein-ligand complex is no
longer stable.
Immunoaffinity chromatography
• Monoclonal antibodies can be attached to the column material.
• The column only binds the protein against which the antibody has
been raised.
• 10,000-fold purification in a single step!
• Disadvantges
– Difficult to produce monoclonal antibodies (expensive $$!)
– Harsh conditions to elute the bound protein
Other chromatographic methods
• Adsorption chromatography - nonpolar molecules physically
adosrbed on the surface of an insoluble substance (alumina,
diatomeceous earth, silica gel, etc.) through Van der Waals forces.
• Molecules eluted from the column by organic solvents (chloroform,
hexane, ethyl ether).
• Based on the partition of polar column material and nonpolar solvent.
• Not used often with proteins.
• Hydroxyapatite chromatography - gels of crystalline hydroxyapatite
(an insoluble form of calcium phosphate) adsorb proteins.
• Separation occurs with a gradient elution of the column with
phosphate buffer.
Other chromatographic methods
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Paper chromatography - separation of small polar molecules. Mostly used to
separate amino acids, oligopeptides. Historically the first chromatography but not really
used today. However, principles of its use are useful to know.
Rates of migration of the substances are determined by relative solubilities in the polar
stationary phase (paper) and the nonpolar mobile phase
A given solute is distributed between the
mobile and stationary phases according to its
partition coefficient
concentration in stationary phase
Kp =
concentration in mobile phase
Molecules are separated according to their
polarities, with nonpolar molecules moving faster
than polar molecules
Other chromatographic methods
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•
After the solvent has migrated an appropriate distance, the chromatogram is removed
from the solvent and dried. If not colored, the separated materials can be detected by
radioactivity, fluroescence, etc.
The migration rate of the substance is expressed by the following ratio:
Distance traveled by substance
R =
•
f has a characteristic
Distance R
traveled
by solvent front
Each substance
f value for a given solvent and paper type.
•
Reverse-phase chromatography (RPC)- separates nonpolar substances including
denatured proteins.
Form of liquid-liquid partition chromatography in which the polar character of the phases
is reversed relative to paper chromatography. Stationary phase is nonpolar and the
mobile phase is a more polar liquid. Used to separate lipids but can also be used for
proteins.
Solvent must be highly nonpolar usually high concentration of organic solvent
(acetonitrile) so it denatures proteins so that the hydrophobic cores can interact with the
matrix.
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Other chromatographic methods
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Hydrophobic interaction chromatography (HIC)- the stationary phase is hydrophillic
(agarose gel) with substituted hydrophobic groups.
Interactions with column are relatively weak and can be used for the separation of
native proteins (not denatured), so proteins are separated based on surface
hydrophobicity.
High performance liquid chromatography (HPLC)- may be based on adsorption, ion
exchange, size exclusion, HIC or RPC but is improved because of the noncompressible
matrix.
Can be made of silica and withstand very high pressures (up to 5000 psi) so flow rates
can be very high.
Advantages of HPLC
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High resolution
Fast
High sensitivity
Can be easily automated
Dialysis
• Dialysis-a process that separates molecules according to size
through the use of semipermeable membranes containing pores of
less than macromolecular dimensions.
• Pores in the membrane allow solvents, salts and small metabolites
to diffuse across but block larger molecules.
• Cellophane (cellulose acetate) most commonly used dialysis
material.
• Usually used to change the solvent in which the protein is dissolved
in.
• Can also be used to concentrate a protein solution by placement in
a polymeric dessicant (PEG) which cannot go through the
membrane but absorbs water through the membrane.
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Figure 6-11
Use of dialysis to separate
small and large molecules.
Page 142
Table 6-4 Purification of Rat Liver
Glucokinase.
Electrophoresis
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The migration of ions in an electric field to separate molecules.
Many forms of electrophoresis-we will focus on polyacrylamide gel
electrophoresis (PAGE).
PAGE techniques are often used determine the purity of proteins.
Page 146
Figure 6-20
Apparatus for slab gel
electrophoresis.
Page 147
Figure 6-21
Diagram of a disc
electrophoresis apparatus.
Sodium dodecyl sulfate (SDS-PAGE)
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•
•
Native protein
Native protein is unfolded by
heating in the presence of mercaptoethanol and SDS.
SDS binds to the protein so that it
stays in solution and denatures.
Large polypeptides bind more SDS
than small polypeptides, so proteins
end up with negative charge in
relation to their size.
Thus, we can separate the proteins
based on their mass.
Heat
+
Reductant
+
SDS
- - -C
- N - - - - Denatured protein
with bound SDS
Page 149
Figure 6-24
SDS-PAGE.
Page 149
Figure 6-25
Logarithmic relationship
between the molecular mass of a protein and its
relative electrophoretic mobility in SDS-PAGE.
Page 148
Figure 6-23
Detection of proteins by
immunoblotting.
Isoelectric focusing
• For looking at proteins without charge, proteins can be treated with 6M
urea (denatures but unlike SDS does not put charges on a protein).
• Thus, a mixture of proteins can be electrophoresed through a solution
having a a stable pH gradient in from the anode to the cathode and a
each protein will migrate to the position in the pH gradient according to
its isoelectric point. This is called isoelectric focusing.
• Ampholytes (amphoteric electrolytes)-low molecular mass (600900D) ooligomers with aliphatic amino and carboxylic acid groups with
a range of isoelectric points. Ampholytes help maintain the pH
gradiennt in the presence of high voltage.
• Can also use gels with immobilized pH gradients -made of
acrylamide derivatives that are covalently linked to ampholytes. Used
with a gradient maker to ensure continuously varied mixture when the
gel is made.
Page 150
Figure 6-26
General formula of the
ampholytes used in isoelectric focusing.
Isoelectric focusing
• 2D-gel electrophoresis is an invalubale tool for proteomics.
• Proteome (like genome) is the total number of all proteins expressed
by a cell or organism, but with an emphasis on their quantitation,
localization, modifications, interactions, and activities, as well as their
identification.
• Individual protein bands froma stained gel can be cut out of a gel,
destained, and and the protein can be eluted from the gel fragment for
identification and characterization using mass spec.
Page 150
Figure 6-27
Two-dimensional (2D) gel
electrophoresis.
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Table 6-1 Isoelectric Points of Several
Common Proteins.
Summary of techniques for protein
purification
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Cell lysis techniques - osmolysis, mechanical disruption-high speed
blender, homogenizer, French press, sonication
Salting out and salting in
Chromatography
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–
–
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Ion exchange
Size exclusion
Affinity
others
Dialysis
Electrophoresis
– SDS PAGE
•
Isoelectric focusing
Page 133
Crystallization
Crystallization
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Crystallization of proteinsdifficult.
Protein must be homogeneous
(e.g. pure)
Supersaturated solution prepared
(10 mg/ml) and allowed to stand
until crystals form.
Use x-ray diffraction to observe the
bonds that hold the 3-D shape of
the protein.
3-D structure of proteins
X-ray source
Single
crystal of
protein
Diffraction pattern
Computational
recombination of
scattered x-rays
Structural model
Electron density map
Figure 8-35
X-Ray diffraction photograph
of a single crystal of sperm whale myoglobin.
Page 241
Figure 8-36a Electron
density maps of proteins.
Page 241
Figure 8-36b Electron
density maps of proteins.
Page 241
Figure 8-36c Electron
density maps of proteins.