2. 2
PRESENTATION OUTLINE
•Protein Purification Scheme
•Protein fractionation
•Chromatography techniques
• Affinity Chromatography (AC)
• Hydrophobic Interaction Chromatography
(HIC)
• Ion Exchange Chromatography (IEC)
• Gel Filtration (GF)
• Capillary electrochromatography (CEC)
• Strategies for Protein Purification Solubility,
Aggregation and Re-folding of Proteins
3. 3
Sample
Separation
technique
Fractionation
Purification is a Multi-Step Procedure.
Is there activity?Set aside
N
o
Combine
Fractionsyes
Monitor purity
Assay total protein
Assay enzyme activity
Pure?
Prepare for analytical technique
yes
N
o
Repeat with another
separation
technique until pure
4. 4
General Protein Purification
Scheme
• Grow cells
in media
(vector+tag)
•Bacteria
Suspension
•Bioreactor
Purification Strategy
Expression
SDS PAGE Assay
Solubility
Aggregation
Recombination
Characterization
Mass Spectroscopy
X-ray Crystallography
Functional Assay
Lyse the cells
(appropriate
buffer)
Centrifuge
Collect the pellet
5. 5
1. Evaluate an assay for the protein of interest
2. Shortlist a method to have a reasonable source for that activity
Set Protein Purification Strategy
6. 6
Preparing the sample
(Crude Extract)
Protein from cells or tissue
Microbial cells
or tissue
Break cells,
Blender,
homogenizer,
sonication,
pressure
osmotic Pellet with intact
cells, organelles,
membranes and
membrane proteins
Supernatant with
Soluble protein
7. 7
• As the column separates the proteins in
the mixture, the “effluent” drips into a
series of fraction tubes that are moving
at a specific rate of speed. These tubes
are called fractions.
• Here we are showing 20 tubes. Fraction
collectors in most labs have about 75-
200 tubes.
• How do we know which fractions contain
protein? Total protein a can be estimated
by taking the absorbance at 280 nm in a
spectrophotometer. Aromatic amino
acids absorb light at this wavelength
causing all proteins to have absorbance
at 280nm. Many fraction collectors
measure the A280 as the column is
running.
Collect fractions.
9. 9
• Total protein a can be estimated by
taking the absorbance at 280 nm in
a spectrophotometer.
• The values can be plotted against
the fraction number in is what is
called an elution profile.
• Notice the peaks on the graph.
These indicate where the fractions
are that contain protein.
Question 1. How do we know
which fractions contain protein?
A280
0 0 0 2 5 2 0 0 0 2 5 8 5 2 0 0 2 5 2
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Fraction
#
A280
Fraction #
Peaks
10. 10
• Enzyme activity can be
determined by performing an
enzyme assay on each fraction
that contains protein.
Which fractions contained the desired protein?
A280
0 0 0 2 5 2 0 0 0 2 5 8 5 2 0 0 2 5 2 0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Fraction
#
A280
Fraction #
Enz. Assay.Enz. Assay.
Fraction
#
11. 11
• Enzyme activity can be
determined by performing an
enzyme assay on each fraction
that contains protein.
• Notice the results of the enzyme
assay. The highest activity
corresponds to one of the peaks.
• Now we can have them discard
tubes that don’t have enzyme
activity.
Which fractions contained the desired enzyme?
A280
0 0 0 2 5 2 0 0 0 2 5 8 5 2 0 0 2 5 2 0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Fraction
#
A280
Fraction #
EnzAssay
Results
12. 12
The Way to Chromatography
• In order to isolate sufficient quantities of
protein, you may need to start with kilogram
quantities of source (i.e. bacteria, tissues,
etc.) These amounts can best be handled
using precipitation methods (e.g.
ammonium sulfate precipitation). Later in
the purification, large columns can be used
to handle gram to milligram quantities.
Amounts handled on gels are typically in
microgram quantities.
13. 13
Property Methods
Solubility Precipitation with
ammonium sulfate
(salting out)*
Size / shape Size-exclusion
chromotography
Isoelectricpoint
(charge)
Ion exhange
chromatography
binding to
small
molecules
Affinity
chromatography
Common methods of protein purification
*Ammonium sulfate precipitation is cheap, easy, and accommodates large sample sizes.
It is commonly one of the first steps in a purification scheme.
• Purification procedures attempt to
maintain the protein in native form.
Although some proteins can be
re-natured, most cannot!
• To purify a protein from a mixture,
biochemists exploit the ways that
individual proteins differ from one
another. They differ in:
• Thermal stability: For most protein
purifications, all steps are carried
out at ~5°C to slow down
degradation processes.
14. 14
Picture of protein gel
with lanes showing
sequential purification
steps
Procedure Fraction
vol
(ml)
Total Prot
(mg)
Activity
(units)
Specific
activity
Units/mg
Crude
cellular
extract
1400 10000 100,000 10
Size-
exclusion
90 400 80,000 200
Ion
exchange
80 100 60,000 600
Note: The type and order of steps are customized for each protein to be
purified. An effective purification step results in a high yield (minimal loss
of enzyme activity) and large purification factor (large increase in specific
activity).
Purification Yield
15. 15
Chromatographic Mode Acronym Separation Principle
Non-interactive modes of liquid chromatography
Size-exclusion chromatography SEC Differences in molecular size
Agarose chromatography (for
DNA) for DNA binding proteins
- Diff. in length and flexibility
Interactive modes of liquid chromatography
Ion-exchange chromatography IEC Electrostatic interactions
Normal-phase chromatography NPC Polar interactions
Reversed-phase chromtography RPC Dispersive interactions*
Hydrophobic interaction
chromatography
HIC Dispersive interactions*
Affinity chromatography AC Biospecific interaction
Metal interaction
chromatography
MIC
Complex w/ an immobilized
metal
Chromatographic Modes of Protein Purification
* Induced dipole – induced dipole interactions
19. 19
Affinity Chromatography
Binding Capacity (mg/ml) medium
12mg of histag proteins (MW= 27kDa)
Depends on Molecular weight
Degree of substitution /ml medium
~15mmol Ni2+
Backpressure ~43psi
Change the guard column filter
20. 20
Biopolymer (phenyl agarose - Binding Surface)
Driving force for hydrophobic adsorption
Water molecules surround the analyte and the
binding surface.
When a hydrophobic region of a biopolymer binds to
the surface of a mildly hydrophobic stationary
phase, hydrophilic water molecules are effectively
released from the surrounding hydrophobic areas
causing a thermodynamically favorable change in
entropy.
Temperature plays a strong role
Ammonium sulfate, by virtue of its good
salting-out properties and high solubility in water is
used as an eluting buffer
Hydrophobic Interaction Chromatography
Hydrophobic region
21. 21
ION –EXCHANGE 1
• First, to determine the
charge on a protein, given its
pI and the pH.
• Ion-exchange column
chromatography separates
proteins on the basis of
charge.
• We will start with 4 proteins.
• pH 7.2
• Positive charged column
60 Kd
Low pI (6)
20 Kd
Low pI (7)
20 Kd
Medium pI (7)
5 Kd
Hi pI (8)
22. 22
pos
• The matrix of an ion exchange
is positively charged.
• What do you think will
happen?pos
pos
pos
pos
pos
pos
Run columnRun column
pos
pos
pos
pos
pos
pos
23. 23
• The matrix of an ion exchange
is positively charged.
• Only the pos charged proteins
run through the pos charged
column. The others “stick” to
the column.
pos
pos
pos
pos
pos
pos
pos
pos
pos
pos
pos
pos
pos
24. 24
Fractogel matrix is a methacrylate resin upon which polyelectrolyte
Chains (or tentacles) have been grafted. (Novagen)
Ion Exchange Chromatography
Globular
Protein
Deformation due to interaction with
conventional ion exchanger
Maintenance of conformation
while interacting with tentacle
ion exchanger
26. 26
• Gel filtration column
chromatography separates
proteins on the basis of size.
• We will start with 4 proteins.
• You will want to purify the
“yellow one”
60 Kd
Low pI (6)
20 Kd
Low pI (7)
20 Kd
Medium pI (7)
5 Kd
Hi pI (8)
Gel Filtration
27. 27
• The matrix of a size-exclusion
chromatography column is
porous beads.
Run columnRun column
28. 28
• The matrix of a gel filtration
column are beads with
pores.
• The large gray proteins
can’t fit in pores so flows
faster.
• The red / yellow medium
sized proteins get trapped in
the pores.
• The black small proteins
stay trapped in pores longer.
30. 30
ATP immobilized on polyacrylamide resin
DNA Binding Proteins
Heparin Sepharose
Negatively charged proteins (pI >7) are not captured/separated effectively.
31. 31
Capillary Electrochromatography
• CEC is an electrokinetic separation technique
• Fused-silica capillaries packed with stationary phase
• Separation based on electro-osmotically driven flow
• Higher selectivity due to the combination of chromatography and
electrophoresis
Fused silica tube filled with porous methacrylamide-stearyl
methacrylate-dimethyldiallyl ammonium chloride monolithic
polymers, 80 x 0.5mm i.d., 5.5kV. High Plate count ~ 400,000
Height Equivalent to a Theoretical Plate /Plate Count (HETP) H = L/N
number of plates N = 16(t/W)2
where L = column length, t = retention time, and W = peak width at baseline
38. 38
Reagent Derivatization Detection
o-Phthaldialdeyhde
Precolumn/
Postcolumn
FL, Ex 340nm/Em 400nm
Fluorescamine
Precolumn/
Postcolumn
FL, Ex 390nm/Em 490nm
Indocyanine greeen Precolumn
FL, Ex 765nm/
Em 820-840nm
Detection of Proteins by Derivatization with Higher Sensitivity
1000 times more sensitive than UV-Vis detection
39. 39
Solubility of a Protein
Membrane proteins
1. Removal of unbroken cells from the cell lysate by low speed centrifugation
(20 min at 10,000 g).
2. Isolation of the membrane particles from the supernatant by
ultracentrifugation (60 min at >100 000 g).
3. Washing of the membrane particle to remove all soluble proteins.
4. Solubilization of protein from the membrane particles by a mild detergent.
(detergent: protein ratio = 1:10)
5. Phosphate buffers(0.1M-0.5M), 5-50% glycerol helps.
• Depends strongly on the composition of the lysis buffer.
• Salt concentration
Freeze-thaw protocol
* Freeze quickly on dry ice and leave for 3 min.
* Thaw immediately at 42 °C. Vortex vigorously to mix well.
* Repeat the two previous steps three more times (4 cycles in all).
40. 40
Protein Aggregation
• Numerous physicochemical stresses can induce protein aggregation:
• Heat, pressure, pH, agitation, freeze-thawing, dehydration, heavy metals,
phenolic compounds, and denaturants.
41. 41
Denaturation and Renaturation
Variables Good starting point
Buffer composition (pH, ionic strength) 50 mM Tris-HCl, pH 7.5
Incubation temperature 30°C
Incubation time 60 min
Concentration of solubilizing agent 6 M guanidine-HCl or 8 M urea
Total protein concentration 1-2 mg/ml
Re-folding of Proteins
The addition of a mixture of reduced and oxidized forms of low molecular
weight thiol reagent usually provides the appropriate redox potential to allow
formation and reshuffling of disulfide bonds
(1-3 mM reduced thiol and a 5:1 to 1:1 ratio of reduced to oxidixed thiol)
The most commonly used are glutathione, cysteine and cysteamine.
Solubilization of Aggregated
Proteins
42. 42
Polyethylene glycol
(PEG 3350)
0.1-0.4 g/L L-Arginine hydrochloride 0.4-0.8M
Nondenaturating
concentrations of Urea
< 2.0 M K-Glutamate ~5M
Nondenaturating
concentrations
Gdm/ClH
< 1.0 M Proline ~1M
Methylurea 1.5-2.5 M Glycerol 20-40 %
Ethylurea 1.0-2.0 M Sorbitol 20-30 %
Formamide 2.5-4.0 M Sucrose ~1M
Methylfomamide 2.0-4.0 M Trehalose ~1M
Acetamide 1.5-2.5 M TMAO
(trimethylamine N-oxide)
~1M
Ethanol Up to 25% Sulfo Betaine ~1M
Reagents used for Re-folding of proteins
43. 43
n-Penthanol 1.0-10.0 mM Lauryl Maltoside 0.06 mg/ml
n-Hexanol 0.1-10.0 mM CETAB 0.6 mg/ml
Cyclohexanol 0.01-10.0 mM CHAPS 10-60 mM
Tris
> 0.4 M Triton X-100 10 mM
Na2SO4 or K2SO4 0.4-0.6 M Dodecyl Maltoside 2.0-5.0 mM
Cyclodextrin 20-100 mM Sarkosyl 0.05-0.5 %
Reagents used for Re-folding of proteins
Cont.
44. 44
6xHis Tagged Protein Detection Directly on the Gel (from
Pierce)
E. coli lysates expressing 6xHis-tagged proteins, stained with the Pierce
6xHis Protein Tag Staining Kit
45. 45
GST•Bind™ Purification Kits
His•Bind® Purification Kits
Magnetight™ Oligo d(T) Beads
MagPrep® Streptavidin Beads
Protein A and Protein G Plus Agaroses
S•Tag™ Purification Kits
Streptavidin Agarose
T7•Tag™ Affinity Purification Kit
ProteoSpin™ CBED (Concentration, Buffer Exchange and Desalting) Maxi
Kit — Effectively desalts and concentrates up to 8 mg of protein with an
efficient, easy-to-use protocol.(Norgen Biotek Corporation)
ProteoSpin™ Detergent Clean-up Micro Kit — Provides a fast and effective
procedure to remove detergents including SDS, Triton® X-100, CHAPS, NP-40
and Tween 20.
Commercially available protein purification kits
This flow chart is more simplified than the other one. What do you think?
For “separation graphic” show image of the chromatographic apparatus instead of a box.
Combine fractions box: Graphic of little tubes pouring into a bigger beaker.
We need to fit in a short text block with a description of the steps in order using numbers to label the steps.
ANIMATION.
Show cartoon of microbial cells or tissue.
Break the cells
Have colors which show cellular membranes and junk contrasting with the proteins into solution.
User clicks the PLOT VALUES button.
Animation: A graph grows.
After the animation ends
The next appears and the user is prompted to discard the fractions that don’t belong by clickin on the tubes.
Go to next screen to see what it might look like.
Should the A280 value table go away? And just leave the profile to “declutter” the screen.
Peter feels strongly that they should not be encouraged to throw away at this point. Perhaps set aside would be a better term, or eliminate altogether.
they are prompted to perfom an enzyme activity assay on the remaining tubes.
They are prompted to click the “Enzyme Assay” button.
Go to next screen to see results.
Show the color of activity tapers off in intensity.
Go to next screen.
Since so few assays use color, Peter would like these to be absorbance values.
Should these link to a Glossary? I don’t think so.
The student should not worry if they don’t understand the techniques at this point. This is just to introduce some terminology in context.
Is the table a good idea? If so, it needs to be more complete.
JB Table is a good idea - we don’t want the students to consider SDS PAGE to be a purification technique, though, so I removed it from the table.
More background content needed? Theory of it?
We need the info on the proteins.
Should the proteins all be the same size?
Students click on “run column”
Go to next slide to see results.
Just a next button?
I might have gotten the charges mixed up. Let’s make sure we get this straight.
We might need a legend (or something of the kind) on every page???
Do we need more content on what is size? How is it measure? MW.
Pick real numbers is Kd correct?
Show them all repeated together.
Should these proteins be more blob like than perfect circles?
Each color represents a different size/charge.
Perhaps we should develop a little “protein” profile next to each blob.
Protein 1.
60 kD
I think that we should make it so that we make one of the proteins the one that the student wants to purify.
In both Gel filtration and Ion exchange, let’s make it so that neither method works.
The final point we can make is that you have to do both separation methods in sequence.