Protein Purification
Protein Purification
• In an organism, any one protein is present as a very small percentage of the total biomolecules. • In order to characterize a protein fully, it is necessary to purify it.
• Many possible strategies to purify a protein. • In general, the more you know about the protein's properties, the better strategy you could design to purify it. • Purification strategy will be of several stages, each one taking advantage of different characteristics of the protein.
Protein Purification Principles
• Define objectives
– Purity, activity and quantity for the final product
• Define properties of target protein and critical
impurities
– to simplify technique selection and optimisation
• Develop analytical assays
– for fast detection of protein activity/recovery and
to work efficiently
• Remove damaging contaminants early
– for example, proteases
Protein Purification Principles
• Use a different technique at each step
– to take advantage of sample characteristics which
can be used for separation (size, charge,
hydrophobicity, ligand specificity)
• Minimize sample handling at every stage
– to avoid lengthy procedures which risk losing
activity/reducing recovery
• Minimize use of additives
– additives may need to be removed in an extra
purification step or may interfere with activity
assays
• Minimize number of steps - KEEP IT SIMPLE!
– extra steps reduce yield and increase time,
combine steps logically
Number of Steps and the Yield at Each Step
Yield (%)
100
80
95% / step
60
90% / step
40
85% / step
20
80% / step
75% / step
20% overall
yield!
0
1
2
3
4
5
6
7
8
Number of steps
5
Protein Purification: Practical Aspects
• You have to choose your source of material carefully
– unicellular organism
– organ of a metazoan (or plant)?
• Where is the enzyme located?
– In cytosol?
– Within membrane-bound organelle?
– Secreted?
• You can use differential centrifugation to do a (crude)
separation of various cellular compartments to get a
starting point.
• If the protein is in a specific organelle, density gradient
centrifugation is usually used to purify the organelle first.
Protein Purification: Practical Aspects
• All cells contain proteases – enzymes that catalyze
hydrolysis of peptide bonds
• Upon breaking cells, these are released into the
extract, where they can degrade the protein you want
to purify
• In order to inhibit proteolysis and denaturation, protein
purification is usually carried out
– In the cold (on ice or in a cold room)
– In the presence of protease inhibitors (small molecules that
inhibit specific proteases)
Starting materials
• Natural source or artificial expression system
– Bacteria, yeast, plants, transgenic animals
• Abundance
• Lysis and clarification procedures
– Native or denaturing conditions
• Selective precipitation
– Streptomycin Sulfate, polyethyleneimine (PEI) and
cetyltrimethylammonium bromide (CTAB) for
RNA/DNA
– Ammonium Sulfate for Proteins
Sample Preparation
General considerations:
• Select extraction procedure according to source and
location of protein
• Use gentle procedures to minimize acidification and
release of proteolytic enzymes
• Work quickly at sub-ambient temperatures
• Use buffer to maintain pH, ionic strength
Goal: To stabilize sample
9
The basic techniques
• Concentration (size)
–
–
–
–
precipitation
ultrafiltration
dialysis
centrifugation
• Chromatography
(size/charge/chemistry)
– ion exchange
– size exclusion
– affinity
• Electrophoresis
(size/charge)
–
–
–
–
"native"
denaturing
isoelectric focusing
2-dimensional
• Immunological
(size/charge/chemistry)
– chromatography
– in situ imaging
– immunoblotting
Three Phase Strategy: An aid in developing the
purification scheme
Achieve final purity.
Remove trace impurities,
structural variants,
aggregates, viruses, etc.
Purity
Remove bulk
impurities
Isolate product,
concentrate, stabilize
Polishing
Intermediate
purification
Capture
Step
12
Non-chromatographic protein
purification techniques
• Ammonium
sulfate precipitation
• Sedimentation (rare)
• Detergent extraction
• Heat treatment (especially for recombinant
thermophile proteins expressed in E. coli)
Protein Precipitation
•
"Salting Out" when enough salt has been added, proteins precipitate
•
cold prevents denaturation
•
collect by filtration or centrifugation
•
redissolved in solution using a buffer with low salt content.
•
works best with divalent anions like sulfate, especially ammonium sulfate
which is highly soluble at ice temperatures
Salting out methods
– Solubility is sensitive to ionic strength.
– Initial “salting in” and then “salting out”
– At high salt, solvent tied up with interacting with
salts so that it is insufficient to solubilize proteins.
– 1st go to maximal salt that the target protein is
soluble, centrifuge and discard pellet.
– Now add just enough salt to bring down the target
protein
– Collect pellet
– Redissolve the pellet in low-salt buffer
Ammonium sulfate precipitation
Example:
• Your protein will remain soluble at 30% (w/v)
ammonium sulfate, but precipitates at 40% ammonium
sulfate.
• Slowly add the salt to your protein extract until it
reaches a concentration of 30%, allow precipitation to
occur, and then centrifuge the solution to remove the
precipitate.
• Take the supernatant, and add ammonium sulfate until
it reaches a concentration of 40%
• Allow precipitation to occur, and then centrifuge the
solution to remove the precipitate.
• Dissolve the precipitate in a buffer and dialyze it to
remove the salt.
Solubilities of several proteins in
ammonium sulfate solutions
Other solubility techniques
• Organic solvents
– Same principle as salting out; taking advantage of
different solubilities. Avoid totally denaturing proteins
• pH
– Proteins have many ionizable groups with range of pKs.
– When net charge of protein is zero, this is the
isoelectric point or pI.
– Proteins are typically least soluble at their pI due to
minimizing charge-charge interactions
• Crystallization
– The solubility methods where proteins are ppt can be
used to grow crystals of proteins. This is only done
when the protein is relatively pure.
Solubility of lactoglobin as a function of
pH at several NaCl concentrations
Solubility of hemoglobin at its pI as a
function of ionic strength and ion type
Buffer Exchanges
• Almost all purification steps will use a buffer
with specific pH and/or ionic strength
• The buffer used impacts the protein's
biophysical characteristics
• Why exchange?
– If you have just precipitated a protein with
ammonium sulfate, you now have that
protein in a high salt environment.
How can you remove salt?
Dialysis
• The protein is put in a bag of cellulose
membranes having small pores of controlled
size.
• Proteins bigger than the pores are retained,
while smaller molecules may diffuse out.
• As the volume of the buffer surrounding the
bag is many times (100-1000x) the volume
within the bag, the smaller molecules can be
effectively removed after several changes of
the outer buffer.
Dialysis
Capture
• Quickly remove most damaging contaminants
• Concentrate, adsorption methods
– Ion Exchange most general
– Affinity chromatography can combine capture,
intermediate and polishing steps
– This step should remove most unwanted
contaminants
Intermediate purification
• Use a different technique
• Affinity chromatography, Hydrophobic interaction
chromatography
• Starting conditions are specific for each technique
– Buffer must be compatible with adsorption
– Can change buffer by dialysis or desalting by GFC
• Adsorption techniques result in small volume
concentrated sample
Polishing
• Final removal of trace contaminants
• Often size exclusion chromatography
– Buffer exchange is a part of the process
– Sample volume always increases need to
start with a concentrated sample
• Sample can be concentrated by
– Precipitation (selective or nonselective)
– Ultrafiltration (dialysis under pressure)
Assays, Quantitation and Documentation
• Assay enzyme activity at every step
•
•
•
•
•
– Contaminants at early stages can mask or inhibit
activity
– Inactivation can occur at high temperatures,
because of proteolysis, oxidation, aggregation, etc.
Assay total protein
Run an SDS gel to visualize specific contaminants
Specific activity is defined as units of enzymatic
activity per unit of total protein Yield can be defined in terms of total protein mass,
and total enzyme units
Goal is a high yield and high specific activity.
Detection
• Spectroscopy
– A280 e 1%280 = 14.5 g-1Lcm-1
• Protein Assay
– Bradford (coomassie)
– Biuret (copper)
– Lowry (modified biuret - phosphomolybdotungstate
mixed
A550
acid reduced by Cu2+ and F,Y,W to form
heteropolymolybdenum blue A575
• Enzyme Assay
Absorption spectra of Trp & Tyr
Beer’s law: A = εcl. Used to estimate protein concentration
Biuret Reaction
• Primarily measure peptide bonds
• Cupric acid react with the peptide
groups of proteins and form a
cupric ion complex
• Generate purplish-violet color with
Amax at 540 nm
• Lowry method
• Low sensitivity
Ninhydrin Reaction
• The most widely used reagents for amino acids, peptides,
and proteins
• Reacts with amino groups
• The major step is oxidative deamination of an amino acid
to CO2, NH3, and an aldehyde containing one less
carbon atom than the original amino acid
• Ninhydrin is simultaneously reduced to ninhydrindantin
• Ninhydrindantin reacts with NH3 and produce purple color
(Amax 570 nm)
• Destroy amino acid or peptide and the material detected
cannot be used for further characterization
Assays
• Enzymatic assays
– PNPP is hydrolyzed to PNP and Pi
– p-Nitrophenyl Phosphate (PNPP) is a non-proteinaceous,
non-specific substrate used to assay protein, alkaline and
acid phosphatases
– Fixed time assay
• Mix enzyme and substrate, react for a fixed time, s
• top the reaction with a strong base,
• read the concentration of PNP at pH>10
– Continuous assay
• Monitor PNP production directly in the spec at ph 8
• SDS page for the distribution of proteins by size.
Purification table
Step
Protein
(mg)
Activity
(U)
Specific
Activity
(U/mg)
Yield
Purification
factor
1
2
3
4
1200
600
200
30
800
600
400
300
0.67
1
2
10
–
75%
67%
75%
–
1.5
2
5
Activity: definition of 1 unit (U) will vary, depending upon enzyme
Specific activity: measure of enzyme’s purity = activity/protein
Yield of a step = Units retained after that step/Units input
(Measure of how well enzyme activity is preserved by the step.)
Purification factor = specific activity after the step/before
(Measure of how effective the step is.)
Purification table
• Yield and purification factor can be
expressed
– for the individual step
or
– as cumulative yield or purification factor,
taking into account all steps up to to that point
• You need to be careful to distinguish
between them.
Example: Purification of Rat Liver Glucokinase
Criteria for purity
When is protein pure or pure enough?
• Homogeneity
– protein complexes?
• Constant specific activity
• Practical: further attempts at purification are futile
since the only material left in the fraction is the
material that actually is responsible for the activity
being assayed.
Characterization of Purified Proteins
- SDS PAGE (both reducing and non-reducing)
- Bioassay (if you have one)
- Total protein determination
- UV spectrophotometry
- CD spectrometry
- Amino acid analysis
- N-terminal (& C-terminal?) sequencing
- HPLC?
- Metal analysis
- Mass spectrometry
- NMR spectrometry & X-ray crystallography*
A. Differential Precipitation
• Precipitation: the process of formation of a solid that
was previously held in solution. The solid is
separated from the solution.
• When NH4 SO4 or polyethylene glycol are added to a
protein solution
• A precipitate forms and it can be separated from the
solution after centrifugation.
• If the concentrations of NH4 SO4 or polyethylene
glycol are increased, more precipitate forms.
38
Density Gradients
• An equilibrium sedimentation experiment can
be set up with linear gradients of sucrose or
glycerol.
• The protein is loaded on top, and
centrifugation is started.
• The advantage of this technique is that the
gradients tend to be rather stable and allow
one to remove the protein of interest from
them.
• The disadvantage is that they are not so
accurate for determination of S.
Zonal ultracentrifugation
Zonal Centrifugation
•
Zonal centrifugation: Mixture to be separated is layered on top of a
gradient (e.g. sucrose or ficoll) increasing concentration down the tube
- can be continuous or discontinuous (layers)
- provides gravitational stability as different species move down tube at
different rates forming separate bands.
– Species are separated by differences in SEDIMENTATION
COEFFICIENT (S) = Rate of movement down tube/Centrifugal
force
– S is increased for particle of LARGER MASS
(because sedimenting force a M(1-vr)
– S is also increased for MORE COMPACT STRUCTURES of equal
particle mass (frictional coefficient is less)
Zonal ultracentrifugation
Isopycnic Centrifugation
•
Isopycnic (equal density) centrifugation: Molecules separated on
EQUILIBRIUM POSITION, NOT by RATES of sedimentation.
•
Each molecule floats or sinks to position where density equals
density of solution (e.g. CsCl gradient for nucleic acid separation).
Gel Filtration Chromatography
•
Gel Permeation Chromatography.
•
Separates protein molecules according to their molecular size.
•
The solution is inserted to the top of a specialized column.
•
This column consists of specialized porous beads.
•
Small molecules of protein enter the beads while large molecules can’t
and stay in the space between the beads.
•
Therefore, large molecules flow more rapidly through the column and
emerge first from the bottom of the column.
•
Advantage: larger quantities of proteins can be separated.
•
Disadvantage: Lower resolution.
45
Sephadex
In Sepadex, the dextran chains are covalently linked to a highly
cross-linked agarose matrix. The figure shows a
schematic of a section through a Sepadex particle
Gel Filtration Chromatography
47
Gel filtration chromatography
Gel filtration chromatography
• Vbed = Vbeads + Vvoid
– The void volume is the volume surrounding the
beads.
– The bed volume is the total volume of the column.
– Vvoid is typically about Vbed /3
• A protein can be characterized by its elution volume
(Velution), which is the volume of solvent required to
elute it from the column. The relative elution volume (=
Velution / Vvoid) is a quantity independent of the particular
column used.
• By standardizing the column with proteins of known
size, one can use this technique to estimate molecular
weight (assuming that the shape is close to that of the
standards – more or less spherical).
Principles of gel chromatography (con’d)
Gel Filtration Elution Volumes as a Function of Molecular Weight
Commonly Used Gel Filtration Materials
Ion exchange chromatography
• This technique uses the charge on a protein to separate
it from other proteins.
• In the process of ion exchange, ions in solution replace
ions that are electrostatically bound to an inert support
carrying groups with the opposite charge.
• Polyelectrolytes, such as proteins, can bind to either
cation or anion exchangers, depending on their net
charge (i.e. depending on the pH).
• The protein can be eluted from the column either by
changing the pH or by increasing the salt concentration,
which shields the charges and thus decreases their
attraction.
• Elution is most often carried out by applying a salt
gradient to the column.
Ion – Exchange Chromatography
•
A cation or an anion is attached to the resin beads.
•
Depending upon the electrical properties of the proteins, they
may attach to the column.
– positively charged proteins will stick to a negatively charged column.
•
These proteins can then be removed by washing the column with
either a strong salt solution or changing the pH of the wash buffer.
•
Anion exchangers such as DEAE ( Diethyl aminoethyl) are used.
•
Attraction of proteins at a pH above the isolectric point of the
protein.
•
Cation exchangers such as CM ( Carboxy methyl) are used.
Attraction of protein at a pH below the isoelectric point of the
protein.
Ion – exchange Chromatography
Ion – exchange Chromatography
Elution
•
Done by washing the column with a strong salt solution (NaCl) which
increases the ionic strength thereby pushing out the proteins.
Typical Gradient Elution
Device for generating a linear concentration gradient.
Ion exchange chromatography
using stepwise elution
Molecular formulas of cellulosebased ion exchangers
Some common ion exchangers.
Ion Exchange Chromatography (con’d)
Hydrophobic interaction chromatography •
Principle
– Proteins are separated by hydrophobic interaction on
columns with hydrophobic groups attached (e.g. phenyl-,
octyl groups)
•
Surface hydrophobicity
– Hydrophobicity of amino acid sidechains
– Tryptofan > Isoleucine, Phenylalanine > Tyrosine >
Leucine > Valine > Methionine
– Most hydrophobic sidechains are buried in interior of protein,
but some (clusters of) hydrophobic groups occur at surface
of protein.
– Surface hydrophobic sidechains can interact with
hydrophobic groups of a column.
Hydrophobic interaction chromatography • Temperature
Increasing temperature --> stronger hydrophobic
interactions
• Sample (application)
Column having high concentration of a salt promotes
binding (for example ammonium sulfate just below
the concentration that starts to precipitate protein).
• Elution of bound proteins
Negative gradient of salting-out ions (from high to low
concentration).
Hydrophobic interaction chromatography • Hydrophobic group bound to solid phase
• Binding
– high salt (increases water surface tension, decreases
available water molecules, increases hydrophobic
interactions)
• Elution
– decrease salt
– add detergent
– decrease polarity
of mobile phase
Hydrophobic Interaction Separation (HIC)
Reversed Phase HPLC
•
Purifies proteins according to their hydrophobicity
•
The stationary phase is hydrophobic and the mobile phase is more
hydrophilic than the stationary phase - “Reversed" from normal phase
chromatography
•
Silica beads with attached hydrocarbon chain groups.
•
The hydrocarbon groups may be octacecyl, butyl, propyl,
phenyldimethyl
•
The hydrophobic groups of the protein will bind to the beads.
•
The hydrophilic proteins will emerge from the column first.
•
Many different sizes of columns with varying widths
Preperative (2.5-5cm) Ä Analytical (0.4-1cm) Ä Microbore (0.10.2cm)
•
The solvents used:
– Aqueous Phase: Water + 0.1% Triflouroacetic acid
– Organic Phase: Acetonitrile
70
“Reversed Phase” Chromatography (RPC)
(elution with organic solvents)
Affinity chromatography
• Takes advantage of the fact that many proteins
specifically bind other molecules as part of their function.
• Construct a column containing the ligand covalently
attached to a matrix.
• Upon passing the protein solution through such a column,
only the proteins that can bind the ligand will be retained
on the column.
• Then the conditions can be adjusted to effect release
from the ligand.
• Often this can be done simply by eluting with the soluble
version of the ligand.
Covalent linking of ligand to agarose
Derivatization of epoxy-activated
agarose
Affinity Chromatography
•
Other proteins can be separated by this method based on their
affinity for specific groups or compounds.
•
Examples:
Antigen
Antibody
Antibody
Antigen
Substrate
Enzyme
Concavalin A
Glycoprotein
Hormone
Binding
Protein/Receptor
Example: purification of staphylococcal
nuclease by affinity chromatography on
bisphosphothymidine-linked agarose
Immuno-Affinity Chromatography
• Antibody fixed to
matrix
• Protein binds to
antibody
• Wash unbound and
loosely bound
proteins off column
• Elute protein with
change in salt/pH
Typical Affinity Separation
Purification schemes
Gel electrophoresis
• General technique to analyze mixtures of proteins, and
for limited purification of proteins.
• The protein is driven through a viscous solvent by an
applied electric field (E), due to the charge of the protein
(z):
v = Ez/f
(f = the protein's frictional coefficient)
• A protein’s electrophoretic mobility (µ) is defined:
µ = v/E
• Typically this is performed in the presence of a gel
support, such as polyacrylamide
– prevents convection currents
– enhances separation by serving as a molecular sieve
Native gel electrophoresis
• The native protein migrates through the gel according to the charge of the protein at the pH of the buffer system.
• Useful for getting relatively pure protein
• Not used very often.
SDS‐polyacrylamide gel electrophoresis
(SDS‐PAGE)
• Most common form of PAGE, but not useful for purification
of proteins in their native conformation.
• Proteins are solubilized with the detergent SDS
(sodium dodecyl sulfate):
– binds polypeptide at a ratio of ~1 SDS per 2 amino acid
residues
– denatures protein → converts to roughly rod-like shape
– protein has a negative charge roughly proportional to its
mass
– The additional negative charge is much greater than the
protein's intrinsic charge, which can usually be ignored.
SDS‐polyacrylamide gel electrophoresis
(SDS‐PAGE)
• As charge/mass ratio is almost constant and the molecular
shapes are all similar, separation is on the basis of size.
• Smaller polypeptides migrate faster and larger ones
migrate slower, due to the gel filtration effect.
• There is an empirical relationship between mobility and
molecular weight:
µ ∝ 1/log Mr
• Average pore size of the gel can be controlled by varying
the concentration of acrylamide before initiating
polymerization.
• Higher percentage polyacrylamide gels (smaller pore
sizes) will result in better resolution of smaller polypeptides.
Polymerization of acrylamide and N,N¢‐methylenebisacrylamide
Electrophoresis
• Tris‐glycine buffer
• 10% SDS
Western blotting
• Separate proteins by electrophoresis
• Transfer to membrane (e.g. nitrocellulose)
• Bind primary antibody
• Bind secondary antibody
• Detection
Isoelectric focusing
• A pH gradient is set up by electrophoresing
polyampholytes (300-600 Da oligomers bearing amino
and carboxylate groups in varying rations) in a gel tube.
• The more basic ones (cationic) will accumulate near the
cathode and the more acidic (anionic) will accumulate
near the anode, thereby establishing a continuous pH
gradient.
• When a protein is applied to the gel, it will migrate toward
the anode if it is negatively charged or toward the
cathode if positively charged, until it reaches the region
corresponding to its pI, where it will stop.
• Proteins are often denatured in 6M urea, which does not
change the protein's charge (unlike SDS).
General formula of the ampholytes used in isoelectric focusing.
Example
• A protein with a pI of 5.2 is introduced near the cathode end, which has pH of 9.5 (in this gel). • It is then negatively charged and will migrate toward the anode.
• As it migrates in this direction, the pH will steadily decrease,
and the amount of negative charge carried by the protein will also decrease, as more and more ionizable groups become protonated. • Thus it will slow down, until it hits the region where pH = 5.2,
where it will experience no further force. • If it diffuses in either direction, it will pick up charge and the electric field will force it back. • This results in the protein being concentrated (focused) in a very narrow region of the gel.
2‐Dimensional Gels
• This is a powerful technique that combines isoelectric focusing with SDS‐PAGE.
• Proteins are separated in the first dimension by isoelectric focusing (IEF).
• Then this tube is attached to the side of an SDS‐
polyacrylamide gel. SDS‐PAGE provides the second dimension. • Each protein migrates to a semi‐unique spot according to its pI and molecular weight (MW).
Two‐dimensional (2D) gel electrophoresis
Using antibodies
Antibodies (immunoglobulins) bind specific antigens/epitopes
monoclonal - all bind same epitope
polyclonal - mixture that binds several epitopes
Secondary antibodies - anit-immunoglobulins (antibodies to antibodies)
Using antibodies