Lecture 7: Protein purification
– Protein purification
Methods of solubilization
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1st step is to get the protein into solution
If the protein is located in the cytosol, we need to break open the cell.
For animal cells this can be accomplished with osmotic lysis. Put the cells
in hypotonic solution; less solutes than inside the cell.
For cells with a cell wall (bacteria, plants) we have to use other methods.
For bacteria, lysozyme is often effective - Selectively degrades bacterial
cell wall.
Can also use detergents or organic solvents, although these may also
denature the protein.
Mechanical processes to break open the
cell
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High speed blender
Homogenzier
French press
Sonicator.
After the cells have been broken, the crude lysate, may be filtered or
centrifuged to remove the particulate cell debris. The protein of interest is
in the supernatant.
For proteins that are components of
membranes or subcellular assembly
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Remove the assembly from the rest of the cellular material (mitochondria
for example).
Can be done by differential centrifugation-cell lysate is centrifuged at a
speed that removes only the cell components denser than the desired
organelle followed by a centrifugation at speed that spins down the
organelle.
Stabilization of proteins
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pH
Temperature
Inhibition of proteases
Retardation of microbes that can destroy proteins
– Sodium azide is often used.
Assay of proteins
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Done throughout the purification process to make sure that your protein of
interest is there.
If the protein of interest is an enzyme, using a reaction for which that
enzyme is a catalyst is a good way to monitor protein recovery.
Monitor the increase of the product of the enzymatic reaction
– Fluorescence
– Generation of acid to be monitored by titration
– Coupled enzymatic reaction - couple with another enzyme to make an
observable substance.
Immunochemical techniques to assay for
proteins
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Use specific immunoglobulins (antibodies), proteins that interact
specifically with the protein of interest and can be easily monitored.
Antibodies are produced by an animal’s immune system in response to the
introduction of a foreign protein.
Antibodies specifically bind to the foreign protein.
Extracted from blood serum of animal that has been immunized against a
particular protein.
Many different antibodies in sera with different specificities and binding
affinities toward the protein of interest.
Immunochemical techniques to assay for
proteins
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Immune cells that produce antibodies normally die after a few cell divisions
so it is difficult to get a specific antibody clone.
We can make monoclonal antibodies by fusing a cell producing the
desired antibody with a cancer cell (myeloma).
– This results in a hybridoma that is essentially an immortal cell, so large
quantities of monoclonal antibody can be produced.
Page 130
Figure 6-1An enzyme-linked immunosorbent
assay (ELISA).
Summary of initial steps of protein
purification
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Choose source of proteins.
Solubilize proteins.
Stabilize proteins.
Specific assay for protein of interest
– Enzymatic activity, immunological activity, physical characteristics (e.g.
molecular mass, spectroscopic properties, etc.), biological activity
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Assay should be:
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Specific
Rapid
Sensitive
Quantitative
Things to monitor during protein
purification
• Things to monitor during protein purification
– Total sample volume
– Total sample protein (est. by A280; 1.4-1.0 mg/ml)
– Units of activity of desired protein (based on specific assay)
• Other basic information to track
– % yield for each purification step
– Specific activity of the desired protein (units/mg of protein)
– Purification enhancement of each step (e.g. “3.5 fold purification”)
• In designing a purification scheme you have to balance purification
with yield.
Characteristic
Procedure
Solubility:
1.
2.
Salting in
Salting out
Ionic charge:
1.
2.
3.
Ion exchange chromatography
Electrophoresis
Isoelectric focusing
Polarity:
1.
2.
3.
Adsorption chromatography
Paper chromatography
Reverse-phase
chromatography
Hydrophobic interaction
chromatography
4.
Molecular size:
1.
2.
3.
4.
Dialysis
Gel electrophoresis
Gel filtration chromatography
Ultracentrifugation
Binding specificity:
1.
Affinity chromatography
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Solubilities of proteins
Multiple acid-base groups on proteins affect their solubility properties.
Solubility of a protein is therefore dependent on concentrations of dissolved
salts, the polarity of solvent, the pH and the temperature.
Certain proteins will precipitate from solutions under conditions which others
remain soluble-so we can use this as an initial purification step of proteins.
Salting out or salting in procedures take advantage of ionic strength
Ionic strength (I) = 1/2c Z
2
i i
Ci = molar concentration of ionic species
Zi = ionic charge
Page 131
Figure 6-2 Solubilities of several proteins in
ammonium sulfate solutions.
Solubilities of proteins
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A protein’s solubility at a given ionic strength varies with the types of ions in solution.
The order of effectiveness of these various ions in influencing protein solubility is
quite similar for different proteins and is due to the ion’s size and hydration.
The solubility of a protein at low ionic strength generally increases with the salt
concentration. This is called salting in. As the salt concentration increases the
additional counterions more effectively shield the protein molecule’s multiple ionic
charges and thereby increase the protein’s solubility.
At high ionic strengths the solubilities of proteins as well as most other substances
decrease. This is called salting out and results from a competition between the
added salt ions and the other dissolved solutes for molecules of solvation.
Page 131
Figure 6-3 Solubility of caboxy-hemoglobin at
its isoelectric point as a function of ionic
strength and ion type.
Ammonium Sulfate
(% saturated)
0
10
20
30
40
50
60
70
80
90
Sample A280
1000
900
600
300
100
75
50
40
25
20
Activity assay (units)
200
200
200
190
170
100
30
5
0
0
Solubilities of proteins
• Salting out is one of the most commonly used protein
purification procedures.
• By adjusting the salt concentration in a solution with a mixture of
proteins to just below the precipitation point of the protein to be
purified, many unwanted proteins can be eliminated from solution.
Then after the precipitate is removed by filtration or centrifugation,
the salt concentration of the remaining solution is increased to
precipitate the desired protein.
• Ammonium sulfate is the most commonly used reagent
– High solubility (3.9 M in water at 0 ºC)
– High ionic strength solution can be made (up to 23.5 in water at 0 ºC)
Note-certain ions (I-, ClO4-, SCN-, Li+, Mg2+, Ca2+ and Ba+) increase the
solubilities of proteins rather than salting out. (also denature proteins).
Solubilities of proteins
• Water-miscible organic solvents also precipitate
proteins.
– Acetone, ethanol
– Low dielectric constants lower the solvating power of
their aqueouse solutions for dissolved ions.
• This technique is done at low temperatures (0 ºC)
because at higher temperatures, the solvent evaporates.
• Can magnify the differences in salting out procedures.
• Some water-miscible organic solvents (DMF, DMSO) are
good at solubilizing proteins (high dielectric constants).
Solubilities of proteins
• Proteins have various ionizable groups (many pK’s)
• At a pH characteristic for each protein, the positive
charges on the molecule exactly balance the negative
charges (isoelectric point, pI).
• At pI, the protein has no net charge and is immobile in
an electric field.
• Therefore, solubility can be influenced by changes in the
pH.
Page 132
Figure 6-4 Solubility of b-lactoglobin as a
function of pH at several NaCl concentrations.
Solubilities of proteins
• A protein in a pH near its isolectric point is not subject to
salting in.
• As the pH is moved away from the pI of the protein, the
protein’s net charge increases and it is easier to salt in.
• Salts inhibit interactions between neighboring molecules
in the protein that promote aggregation and precipitation.
• pI’s of proteins can be used to precipitate proteins.
Page 133
Table 6-1 Isoelectric Points of Several
Common Proteins.
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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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
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+
Positively
charged (basic)
protein or enzyme
CM cellulose
cation exchanger
If these groups are basic in nature, they interact with negatively charged
molecules 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
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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.
Page 134
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.
Page 137
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
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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