Protein purification
1-Ion exchange chromatography
•
•
Ion exchange contain charged groups.
If these groups are acidic in nature they interact with positively charged proteins and
are called cation exchangers.
CM cellulose
cation
exchanger
•
+
CH2-COO
+
+
CH -COO2
+
Positively
charged (basic)
protein or enzyme
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)
Diethylaminoethyl (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-+
Carboxymethel (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
• 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
.
• Cation exchangers bind to proteins with positive
charges.
• Anion exchangers bind to proteins with negative
charges.
Page 134
Ion exchange chromatography
using stepwise elution.
•
•
•
•
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.
2-Size Exclusion Chromatography (SEC)
Gel Permeation chromatography(GPC)
Gel Filtration Chromatography (GFC)].
• In SEC, there is no interaction between the sample
compounds and the column packing material.
Instead, molecules diffuse into pores of a porous
medium. Depending on their size relative to the pore
size, molecules are separated. Molecules larger than
the pore opening do not diffuse into the particles,
while molecules smaller than the pore opening enter
the particle and are separated. Large molecules elute
first. Smaller molecules elute later
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
Page 137
Gel filtration chromatography.
3-Affinity chromatography
• Many proteins can bind specific molecules very
tightly but noncovalently.
• We can use this to our advantage with affinity
chromatography.
Affinity chromatography
•
•
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.
• purification in a single step!
• Disadvantges
– Difficult to produce monoclonal antibodies (expensive $$!)
– Harsh conditions to elute the bound protein
– E.g used to separate factor VIII
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.
• .
Page 139
Figure 6-11
Use of dialysis to separate
small and large molecules.