Lecture 7: Protein purification

Protein purification
 Methods of solubilization
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
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
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

pH
Temperature
Inhibition of proteases
Retardation of microbes that can destroy proteins
Sodium azide is often used.
 Assay of proteins
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
Use specific immunoglobulins (antibodies), proteins that interact specifically with the protein of interest and
can be easily monitored.
Antibodies are produced by an animals 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
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.
 Figure 6-1An enzyme-linked immunosorbent
assay (ELISA).
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 Summary of initial steps of protein
purification
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
Assay should be:
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. Salting in
2. Salting out
Ionic charge: 1. Ion exchange chromatography
2. Electrophoresis
3. Isoelectric focusing

Polarity: 1. Adsorption chromatography

2. Paper chromatography
3. Reverse-phase chromatography
4. Hydrophobic interaction
chromatography

Molecular size: 1. Dialysis

2. Gel electrophoresis
3. Gel filtration chromatography
4. Ultracentrifugation
Binding specificity: 1. Affinity chromatography
 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

2
Ionic strength (I) = 1/2c Z
i i
Ci = molar concentration of ionic species
Zi = ionic charge
 Figure 6-2 Solubilities of several proteins in
ammonium sulfate solutions.
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 Solubilities of proteins
A proteins 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 ions 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 molecules multiple ionic
charges and thereby increase the proteins 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.
 Figure 6-3 Solubility of caboxy-hemoglobin at
its isoelectric point as a function of ionic
strength and ion type.
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Ammonium Sulfate 0 10 20 30 40 50 60 70 80 90
(% saturated)

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 C)
High ionic strength solution can be made (up to 23.5 in water at 0 C)

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 C)
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 pKs)
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.
 Figure 6-4 Solubility of -lactoglobin as a
function of pH at several NaCl concentrations.
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 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
proteins net charge increases and it is easier to salt in.
Salts inhibit interactions between neighboring molecules
in the protein that promote aggregation and precipitation.
pIs of proteins can be used to precipitate proteins.
 Table 6-1 Isoelectric Points of Several
Common Proteins.
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 Column chromatography
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
Ion exchange
Hydrophobic
Gel filtration
Affinity
 Ion exchange chromatography
Ion exchange resins contain charged groups.
If these groups are acidic in nature they interact with positively charged proteins and are called cation exchangers.

+ Positively
-
CH2-COO + charged (basic)
-
If these groups are basic in nature, they interact with negatively
+
charged molecules and are called anion
protein or enzyme
exchangers. CH2-COO
+
CM cellulose
cation exchanger

Negatively
charged (acidic)
CH2-CH2 -NH+(CH2CH2) - -
protein or enzyme
CH2-CH2 -NH+(CH2CH2) -
-
DEAE cellulose
anion exchanger
 Ion exchange chromatography
For protein binding, the pH is fixed (usually near neutral) under low salt
conditions. Example cation exchange column

+ Positively charged protein

CH2-COO- + or enzyme bind to the
+ column
CH2-COO- +
CM cellulose
cation exchanger -
-
-
- 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 Cl-
will interact with our positively charged protein to elute off the column.

-+ + + Increasing
CH2-COO [NaCl] of the
- +
CH2-COO + elution buffer
CM cellulose
cation exchanger

- Na Na+2
+ Cl-
CH2-COO Cl- +
- Na+ +
CH2-COO Cl-

- +
+
CM cellulose Na+2 Cl
cation exchanger
 Ion exchange chromatography
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.
 Figure 6-6 Ion exchange chromatography using
stepwise elution.
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 Ion exchange chromatography
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.
 Figure 6-7 Device for generating a linear
concentration gradient.
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c = c2 - (c2 - c1)f
 Figure 6-8 Molecular formulas of cellulose-
based ion exchangers.
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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 = V x + 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.
 Figure 6-9 Gel filtration chromatography.
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 Figure 6-10 Molecular mass determination
by gel filtration chromatography.
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 Table 6-3 Some Commonly Used Gel
Filtration Materials.
Page 138
 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
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
How does it work?
Ligand - a molecule that specifically binds to the protein of interest.

Inert support + +

Spacer arms Ligand

Affinity material
Inert support
prepared
 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