MIC/BIO/BCH522
Spring 2006
Protein Purification
or
“How not to waste good clean thoughts on dirty enzymes”
A. Kornberg
Scheme
1.
2.
3.
4.
5.
6.
7.
8.
9.
Source of the protein
Lysis Methods
Ammonium Sulfate fractionation
Dialysis
Gel Filtration
Ion exchange chromatography
Protein storage
Assays for activity
Assays for contamination
– Nuclease
– Nucleic Acid
– Nucleoside triphosphates
Initial issues to consider……
1. What type of protein will be expressed?
- prokaryotic origin, the obvious choice is to use E. coli.
- if the protein is from a eukaryotic source, the method of choice will depend on protein solubility,
post-translational modification and codon usage
2. Is the protein expressed in E. coli soluble?
- many eukaryotic proteins don't fold properly in E. coli and form insoluble aggregates (inclusion
bodies).
- is possible to resolubilize the protein from the inclusion bodies or express at a lower temperature.
- a fusion protein with a highly soluble partner such as glutathione-S-transferase (GST), maltose
binding protein (MBP)
3. Does the protein require post-translational modifications for structure/activity?
- complex modifications, like N- and O-glycosylation, phosphorylation, are carried out exclusively by
eukaryotic cells.
4. What is the codon usage in the protein?
- Not all of the 61 mRNA codons are used equally
- major codons in highly expressed genes; minor or rare codons tend to be in genes expressed at a
low level.
- Which of the 61 codons are the rare ones depends strongly on the organism.
- The codon usage per organism can be found in the Codon Usage Database
- Usually, the frequency of the codon usage reflects the abundance of their cognate tRNAs.
- The following problems are often encountered:
Interrupted translation (truncations); Frame shifting; Misincorporation of amino acids
Expression system
1. Escherichia coli
–
–
–
The expression of proteins in E. coli is the easiest, quickest and cheapest method.
There are many commercial and non-commercial expression vectors available with different
N- and C-terminal tags and many different strains which are optimized for special
applications
Vectors which do not add tags are also available with strong promoters
2. Eukaryotic cells
– Human cells
– Xenopus
– Baculovirus infected insect cells
• Insect cells are a higher eukaryotic system than yeast and are able to carry out more
complex post-translational modifications than yeast or E.coli.
• They also have the best machinery for the folding of mammalian proteins and,
therefore, give you the best chance of obtaining soluble protein when you want to
express a protein of mammalian origin.
• The disadvantages of insect cells are the higher costs and the longer duration before
you get protein (usually 2 weeks).
– Yeast
• has some advantages and disadvantages over E. coli.
• One of the major advantages is that yeast cultures can be grown to very high
densities, which makes them especially useful for the production of isotope labeled
protein for NMR.
• The two most used yeast strains are Saccharomyces cerevisiae and the methylotrophic
yeast Pichia pastoris.
Comparison of expression systems
E. coli
Yeast
Insect cells
Mammalian cells
rapid (30 min)
rapid (90 min)
slow (18-24 h)
slow (24 h)
minimum
minimum
complex
complex
Cost of growth
medium
low
low
high
high
Expression level
high
low - high
low - high
low - moderate
secretion to
periplasm
secretion to medium
secretion to medium
secretion to medium
refolding can be
required
refolding may be
required
proper folding
proper folding
none
high mannose
simple, no sialic acid
complex
O-linked
glycosylation
no
yes
yes
yes
Phosphorylation
no
yes
yes
yes
Acetylation
no
yes
yes
yes
Acylation
no
yes
yes
yes
gammaCarboxylation
no
no
no
yes
Characteristics
Cell growth
Complexity of growth
medium
Extracellular
expression
Posttranslational
modifications
Protein folding
N-linked glycosylation
Vectors to use in E. coli
Promoter
source
regulation
induction
Expression level
lac
E. coli
lacI, lacIq
IPTG
low
tac (hybrid)
E. coli
lacI, lacIq
IPTG
moderately high
trc (hybrid)
E. coli
lacI, lacIq
IPTG
moderately high
araBAD
E. coli
araC
l-arabinose
variable
pL
λ
l cI857 (ts)
shift to 42°C
moderately high
T7-lac operator
T7
lacIq
IPTG
very high
Cell growth and expression
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Often, the plasmid must be maintained in a cloning strain and transformed into the expression strain just before use
Following transformation, several individual colonies should be picked and expression verified using SDS-PAGE
The most promising isolate should be used to generate a starter culture. This culture can be used to inoculate a
larger culture in which expression will be induced
The starter culture can be grown in the presence of 0.2 – 0.4% glucose if the promoter is IPTG induced
Typically, induction cultures will contain one or more antibiotics
The use of ampicillin requires special care. The selectable marker, β-lactamase, is secreted into the medium where it
hydrolyzes all of the ampicillin. This point is already reached when the culture is barely turbid. From here on, cells that
lack the plasmid will not be killed and could overgrow the culture
Some possible solutions are:
– grow overnight cultures at 30°C or lower.
– spin overnight cultures and resuspend the pellet in fresh medium to remove β-lactamase.
– use the more stable carbenicillin instead of ampicillin
– spike the cultures with ampicillin at the point of induction.
The induction culture must be aerated as well as possible – this is CRITICAL!! For good aeration, don't use more
medium than 20% of the total flask volume.
Inoculation of the main culture and incubation until OD600 reaches 0.4-1. The optimal OD value depends on the
culture method and the medium. For flask cultures using LB-medium an OD600 of 0.6 is recommended. To increase
the growth rate, we carry out the cultures at 37°C until the OD for induction is reached.
10
OD600
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•
IPTG
1
OD600 = 0.4 to 0.6
0.1
0
2
4
6
8
Time (hours)
10
12
14
Protein purification table
Good record keeping is absolutely essential - monitor protein by SDS-PAGE and OD280
– the amount of protein, the level of contamination and where possible,
Specific activity should be determined at each stage
Total
protein
(mg)
Total
activity
(units)
550
5500
6600
204
102
1020
37
18.7
6
Phenyl
Sepharose
Step
OD260
(x dil)
OD280
(xdil)
Crude cell lysate
1200
30-70% Am.
Sulf. cut
DEAE Sephadex
Specific
activity
(units/m
g)
Purification
% Yield
1.2
NA
NA
5910
5.8
4.8
89.5
187
5070
27.1
4.7
85.8
10.2
102
4420
43.3
1.6
87.2
3.8
5.6
56
3930
70.2
1.6
88.9
Gel Filtration
1.8
3.2
32
2970
92.8
1.3
75.6
Affinity resin
type #1
0.25
0.58
5.8
2520
434.5
4.7
84.8
Affinity resin
type #2
0.27
0.53
5.3
2390
450.9
1
94.8
CM Sephadex
Lysis methods for E. coli
Physical methods:
•
Sonication.
– the most popular technique for lysing small quantities of cells (1-6 L of cell culture).
– Cells are lysed by liquid shear and cavitation.
– DNA is also sheared during sonication,
– The main problem is controlling the temperature addressed by:
• keeping the suspension on ice
• using a number of short pulses (5-10 sec) with pauses (10-30 sec) to re-establish a low temperature.
•
Homogenization.
– Homogenizers are the most common devices to lyse bacteria.
– cells are lysed by pressurizing the cell suspension and suddenly releasing the pressure. This creates a liquid
shear capable of lysing cells.
– the French press ; uses 6000-10,000 psi. Multiple (2-3) passes are generally required to achieve adequate lysis.
•
Freezing and grinding.
–
An alternative lysis method is to freeze the cells directly in liquid nitrogen and ground the frozen cells to a
powder using a mortar and pestle that are chilled with liquid nitrogen. The powder can be stored indefinitely at 80°C and the cell lysate can be prepared by adding the powder to 5 volumes of buffer.
Enzymatic and detergent lysis:
•
Enzymatic lysis
– based on the digestion of the peptidoglycan layer of the bacterial cell wall by lysosyme.
– Gram-negative bacteria, however, have an outer membrane that is external to the cell wall and needs to be
permeabilized to expose the peptidoglycan layer.
– Tris, often used as a buffer in lysis methods, effectively permeabilizes outer membranes.
– Solubilization is enhanced by the addition of EDTA (1 mM).
•
Detergents
Non ionic or zwitterioninc
sodium deoxycholate, brij-35, CHAPS, Triton x-100
DNA removal
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Most lysis methods cause the release of nucleic acids – both DNA and RNA
These have to be removed because they can cause viscosity problems or they can interfere with
subsequent chromatographic steps.
Different methods exists:
Enzymatic digestion by the addition of DNase I (1 µg/ml) to the cell lysate. The mixture is
incubated on ice for 10-15 min.
Mechanical breakdown by shearing during sonication.
When the French Pressure Cell is used it is advisable to add DNase I to the cell suspension.
Nucleic acids can sometimes be readily removed from the sample by the addition of large
cationic compounds such as polyethyleneimine, or streptomycin sulfate.
The nucleic acids bind to these compounds via electrostatic interactions and the complex
precipitates and can be removed via centrifugation.
Add the precipitants to the cell lysate and incubate the solution for 30 min at 4°C.
Precipitation by treatment with polyethyleneimine (0.1% (w/v)) or protamine sulphate (1%
(w/v)) followed by centrifugation.
Often, the protein of interest will also be precipitated using these cationic agents and therefore
must be extracted from the resulting pellet
Protein precipitation methods
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Protein stability in solution dependent on:
– electrostatic interactions (ionogenic, amino acid side chains; salts)
– H-bridges (side chains; water)
– hydrophobic interactions
Perturbation of interactions might cause precipitation of proteins:
– temperature
– pH (dependent on pI)
– salts
– lipophylic agents (e.g. ethanol)
– cross-linking agents (e.g. protamine sulfate)
– water-extracting agents (e.g. polyethylene glycol)
Different proteins precipitate under different conditions
Î FRACTIONATION
Polyethylene glycol
– neutral, non-denaturating compound
– low heat of solution steric exclusion mechanism (binds water)
Protamine sulfate
– small, basic proteins from sperm (large #’s of Arg and Lys residues)
– precipitation of large protein complexes (ribosomes), DNA, RNA by complexation
– precipitation is concentration dependent
Apolar solvents (acetone, alcohol) and Trichloroacetic acid
– unfolds/denaturates proteins
Ammonium sulfate
Why ammonium sulfate ?
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Most proteins precipitate in saturated solution (4 M)
Low heat of solubilization (Æ prevents denaturation of proteins)
Low density of saturated solutions (1.25 g/cm3) Æ proteins can be collected in pellets by
centrifugation
Concentrated solutions prevent microbial growth
Protects most proteins from denaturation
Cheap
•
Applications of ammonium sulfate precipitation
– Concentration of proteins by bulk precipitation
– Purification of proteins by fractionation due to differences in solubility
•
Ammonium sulfate can be added
– as a solid: specific amounts to be added to reach a saturation level
– from a saturated (= 100%, w/v) solution
• Note: saturation level is temperature dependent !!
Ammonium sulfate precipitation theory
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The solubility of protein depends on, among other things, the salt concentration in the solution.
At low concentrations, the presence of salt stabilizes the various charged groups on a protein molecule, thus attracting
protein into the solution and enhancing the solubility of protein. This is commonly known as salting-in.
as the salt concentration is increased, a point of maximum protein solubility is usually reached. Further increase in the
salt concentration implies that there is less and less water available to solubilize protein. Finally, protein starts to
precipitate when there are not sufficient water molecules to interact with protein molecules. This phenomenon of protein
precipitation in the presence of excess salt is known as salting-out.
Many types of salts have been employed to effect protein separation and purification through salting-out.
Of these salts, ammonium sulfate has been the most widely used chemical because it has high solubility and is relatively
inexpensive.
There are two major salting-out procedures:
–
In the first procedure, either a saturated salt solution or powdered salt crystals are slowly added to the protein
mixture to bring up the salt concentration of the mixture.
– The precipitated protein is collected and categorized according to the concentration of the salt solution at which it is
formed. This partial collection of the separated product is called fractionation.
– The protein fractions collected during the earlier stages of salt addition are less soluble in the salt solution than the
fractions collected later.
Whereas the first method just described uses increasing salt concentrations, the following alternative method uses
decreasing salt concentrations.
In this alternative method, as much protein as possible is first precipitated with a concentrated salt solution. Then a series
of cold (near 0ºC) ammonium sulfate solutions of decreasing concentrations are employed to extract selectively the
protein components that are the most soluble at higher ammonium sulfate concentrations. The extracted protein is
recrystallized and thus recovered by gradually warming the cold solution to room temperature. This method has the
added advantages that the extraction media may be buffered or stabilizing agents be added to retain the maximum
enzyme activity. The efficiency of recovery typically ranges from 30 to 90%, depending on the protein. The
recrystallization of protein upon transferring the extract to room temperature may occur immediately or may sometimes
take many hours. Nevertheless, very rarely does recrystallization fail to occur. The presence of fine crystals in a solution
can be visually detected from the turbidity.
Dialysis
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After an ammonium sulfate precipitation step, or an ion exchange chromatography step, the protein of interest may be
in a high salt buffer; salt may hinder the next purification step.
One of the most common methods to remove salt is that of dialysis
The method of dialysis makes use of semi-permeable membranes.
The main feature of this membrane is that it is porous - the pore size is such that small salt ions can freely pass
through the membrane, larger protein molecules cannot (i.e. they are retained). Thus, dialysis membranes are
characterized by the molecular mass of the smallest typical globular protein which it will retain.
This is commonly referred to as the cutoff of the tubing (e.g. Spectrapore #6 dialysis tubing has a cutoff of 1,000
Daltons, meaning that a 1,000 Dalton protein will be retained by the tubing but that smaller molecular mass solutes will
pass through the tubing)
Dialysis proceeds by placing a high salt sample in dialysis tubing (i.e. the dialysis "bag") and putting it into the desired
low salt buffer:
One consequence of dialysis to watch out for is that while salt ions are moving out of the bag, water molecules are
moving into the bag – thus the bag will swell (and protein concentration will decrease)
In the extreme case, the bag may actually swell to the point of rupture.
it is a good idea not to fill the bag completely, but leave a void/space to allow for potential swelling.
For how long and against what volume do you dialyze
samples?
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A useful rule of thumb is that for most types of dialysis tubing the dialysis is 80% complete after four hours
At equilibrium the salt concentration of the sample can be calculated as follows:
•
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The buffer volume for dialysis is a function of the required final concentration of salt in the sample
Dialysis example
– a 10ml protein sample from an ion exchange column elution pool which contains 1.0M NaCl.
– the next step in the purification requires no more than 1mM NaCl in the sample.
–
–
–
–
–
Note that in the above example this would commonly be referred to as a "1:1,000" dialysis.
However, the same results can be achieved with two sequential "1:32" dialyses (i.e. the square root of the
1:1,000 dialysis - in other words, two sequential 1:32 dialyses is equivalent to a single 1:1,000 dialysis):
First dialysis versus 310 ml of buffer: sample NaCl conc will be (10*1.0)/(320) = 31 mM
Second dialysis versus 310 ml of buffer: sample NaCl conc will be (10*0.031)/(320) = 0.97 mM
Thus, instead of making 10 L of buffer, we could make only 620 ml and achieve the same results with two
dialysis steps
Concentration of samples
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Frequently, a particular step will dilute the protein sample
One simple method to concentrate samples is to use a semi-permeable membrane (dialysis bag)
Here, the sample in a dialysis bag is coated with a high molecular weight solute which can readily
be dissolved by the buffer.
– - polyethylene glycols and polyvinyl pyrolidones, MW = 20,000 Da.
If our sample in a dialysis bag is coated with dry forms of the above polymers, water will be
removed from the dialysis bag and hydrate the polymer on the exterior
The result is a decrease in volume of buffer in the dialysis bag,
• i.e., the protein becomes concentrated
Following concentration using a dialysis bag, the bag has to be rinsed with dH20 to remove
excess polymer and re-dialyzed to ensure the buffer is equilibrated to the next purification step
Protein can be concentrated in a variety of other ways
– Amicon spin columns
– Ammonium sulfate precipitation (if the concentration is not too low)
– Certain ion exchange columns can achieve significant concentration, i.e., MonoQ
Chromatography
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Usually several different chromatographic steps are performed.
There is no set order, but a typical order might be cation exchange, gel filtration, and then anion
exchange.
i) Gel filtration - separation on the basis of size
– The matrix contains pores that allow smaller molecules to enter but excludes larger molecules.
– larger molecules spend less time in the matrix and elute first.
– The size of the protein can be estimated from the elution time or volume.
ii) Affinity
– resin contains a specific group (i.e. ligand or antibody) that causes the protein of interest to bind
– ligand affinity, elution can be accomplished by the addition of excess ligand.
– antibody based affinity columns the protein-antibody binding must be weakened by changes of the
pH and/or the salt concentration.
iii) Ion exchange
– The protein sticks to these resins by electrostatic interactions.
– Resins contain either negative (cation exchange resins) or positive (anion exchange resins)
charges.
– In very general terms a protein will stick to a cation exchange resin below its pI and to an anion
exchange resin above its pI.
– what really matters is the local charge distribution on the surface of the protein (i.e. patches of
residues with similar charges).
– Elution of proteins from ion exchange resins involves either a change in pH that results in a change
in the charge of the protein or by increasing the salt concentration.
– Increasing salt provides additional ions than compete with the protein for binding sites on the resin.
Gel filtration
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Gel filtration does not rely on any chemical interaction with
the protein, rather it is based on a physical property of the
protein - that being the effective molecular radius (which
relates to mass for most typical globular proteins).
Gel filtration resin can be thought of as beads which
contain pores of a defined size range.
Large proteins which cannot enter these pores pass
around the outside of the beads.
Smaller proteins which can enter the pores of the beads
have a longer, tortuous path before they exit the bead.
Thus, a sample of proteins passing through a gel filtration
column will separate based on molecular size: large ones
will elute first and the smallest will elute last (and "middle"
sized proteins will elute in the middle).
There are two extremes in the separation profile of a gel
filtration column.
There is a critical molecular mass (large mass) which will
be completely excluded from the gel filtration beads. All
solutes in the sample which are equal to, or larger, than
this critical size will behave identically: they will all eluted in
the excluded volume of the column
There is a critical molecular mass (small mass) which will
be completely included within the pores of the gel
filtration beads. All solutes in the sample which are equal
to, or smaller, than this critical size will behave identically:
they will all eluted in the included volume of the column
Solutes between these two ranges of molecular mass will
elute between the excluded and included volumes
Elution of proteins from a gel filtration column
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If the total column volume is Vt and Vx is the
volume occupied by the resin, then the volume
surrounding the resin is Vo:
Vt = Vx + Vo
The elution volume, Ve, is the volume of solvent
required to elute the protein. The molecular weight
of the protein can be obtained from the following
formula:
Ve/Vo = a*log(Mr) + b
Where "a" and "b" are constants obtained by
calibration of the gel filtration column.
Note that the native molecular weight is obtained,
e.g. hemoglobin would give a measured molecular
weight of 62 kDa and myoglobin (Mr = 17 kDa)
would elute later.
Protein
Myoglobin
TIM (triosephosphate isomerase)
Hemoglobin
IgG (immunoglobulin G)
ATCase (aspartate transcarbamoylase)
Native Mr (Da)
17,200
53,300
62,000
140,000
307,900
#Subunits
1
2
4
4
12
Ion exchange
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Ion exchange resins contain charged groups.
These may be acidic in nature (in which case the resin is a cation exchanger)
or basic (in which case it is an anion exchanger).
Cation and anion exchangers may be broken down further into weak and strong exchangers (reflecting binding
affinity).
Usually, samples are loaded under low salt conditions and bound material is eluted using increasing salt
concentration.
Proteins bound to ion exchange resins are bound via non-covalent ionic (salt-bridge) interactions.
Generally speaking, a protein will bind to a cation exchange resin if the buffer pH is lower than the isoelectric
point (pI) of the protein, and will bind to an anion exchange resin if the pH is higher than the pI.
There are two general types of methods when eluting with a salt solution:
– Gradient elution
– Step elution
A gradient elution refers to a smooth transition of salt concentration (from low to high) in the elution buffer.
Weakly binding proteins elute first, and stronger binding proteins elute last (i.e. they require higher salt
concentrations in the buffer to compete them off the column)
A gradient salt concentration can be made using a gradient maker.
Elution profiles
Linear gradient
Step-wise elution
•If we know the concentration range of salt over which a protein of interest will elute we can simply elute with a buffer
containing that concentration of salt. This is known as a step elution.
•Step elutions are generally faster to run, and elute the protein in a smaller overall volume than with gradient elution.
•They generally work best when contaminants elute at a significantly different salt concentration than the protein of
interest
A typical ion exchange chromatogram
Linear gradient
Resolving peaks and pooling for purity
A.
Panel A:
•
Contaminating peaks will not necessarily be
completely separated from the peak which contains
our protein of interest
•
In the following picture there are two components
being resolved, and they are present in equimolar
amounts (thus, the starting purity is 50%). The yield
and purity are listed for the situation where we were
to pool each peak by splitting at the midpoint
between them (in this particular example the yield
and purity are identical in each case)
•
This gives you some idea of the amount of crosscontamination in each peak as a function of their
separation from one another.
Panel B
•
These are all the same chromatogram, however, we
can pool them differently to get better purity (at the
expense of yield
•
The blue peak is the peak of interest and it is not
resolved from a contaminating peak (in red).
•
The vertical line represents the left-most fraction we
use to pool the peak (we pool all fractions to the
right of the vertical line to get our protein of interest)
•
In the last panel we see that we can achieve about
98.8% purity if we are willing to part with half our
protein!
B.
Storage of proteins
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After you have invested all the hard work to express and purify your target protein, you should not forget to think
about how you want to store your purified protein.
The method of choice depends completely on the stability of the protein and on when and how you want to use it
later on.
For short term storage (up to 24 h), most proteins can be kept at 4°C.
For long term storage (more than a week), it becomes necessary to freeze the protein preparation
Protein preparations will be stable for several years at -80°C or even in liquid nitrogen.
For some proteins, it is important to freeze it rapidly using liquid nitrogen or a dry ice/ethanol mixture to avoid
denaturation.
It is also important to freeze the solution in small aliquots to avoid repeated freezing and thawing which may
reduce the biological activity or affect the structure.
Several stabilizing agents can be added, such as glycerol (5-50% (w/v)), serum albumin (10 mg/ml), reducing
agents (such as 1 mM DTT), and salt (i.e., NaCl, KCl at 20-150 mM, depending on the protein)
To transfer the protein preparation into the storage buffer:
– Add an equal volume of pure glycerol to the protein preparation.
– Dialyze the protein preparation against the storage buffer containing 50% glycerol. This method has the
additional advantage that it results in an approx. threefold concentration.
•
Alternative methods are:
– Storage of the protein at 4°C as an ammonium sulfate suspension.
– Storage of the protein at 4°C or lower in a lyophilized form.
– For the lyophilization it is necessary that the protein is dissolved in a volatile buffer (such as
trimethylamine/HCl; pH range 6.8-8.8).
– Note that not all proteins are stable during the freeze-drying process.
Concentration determination
•
Several methods exist,
– UV Absorbance (280 nm)
– Bradford Assay
– BCA (PIERCE), (modified Lowry)
– Lowry
•
Most protein assays utilizes an internal standard (e.g., BSA or lysozyme) to generate a reference or standard
curve. The protein of choice is then measured against the standard curve. The protein used for the standard
curve will affect the concentration of your purified protein sometimes by several orders of magnitude
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Optimal method is to use the extinction coefficient of the purified protein
Absorption of radiation in the near UV by proteins depends on the Tyr and Trp content (and to a very small extent
on the amount of Phe and disulfide bonds).
Therefore the A 280 varies greatly between different proteins; for a 1 mg/mL solution, from 0 up to 4 for some
tyrosine-rich wool proteins, although most values are in the range 0.5-1.5 (Kirschenbaum, D. M. (1975) Molar
absorptivity and A1%/1 cm values for proteins at selected wavelengths of the ultraviolet and visible regions. Anal.
Biochem. 68, 465-484).
The advantages of this method is that it is simple, and the sample is recoverable. The method has some
disadvantages, including interference from other chromophores, and the specific absorption value for a given
protein must be determined.
The extinction of nucleic acid in the 280-nm region may be as much as 10 times that of protein at their same
wavelength, and hence, a few percent of nucleic acid can greatly influence the absorption.
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Can also provide insight into the degree of aggregation of the protein preparation (A 340 )
RuvA and SSB – two homotetramers
RuvA
SSB protein
1.0
ε = 5.55 x 103 M-1 cm-1
Absorbance (a.u.)
Absorbance (a.u.)
0.20
0.15
OD280 = 0.144
OD260 = 0.088
OD340 = 0.013
0.10
0.05
0.00
250
280
310
340
Wavelength (nm)
370
400
OD280 = 0.755 OD260 = 0.41
OD280 = 0.510 OD260 = 0.26
OD280 = 0.180 OD260 = 0.11
0.5
ε = 3 x 104 M-1 cm-1
0.0
250
280
310
340
Wavelength (nm)
370
400
Final gel from an EcoR124I preparation
A
B
1
2
3
4
100
209
124
80
49
R-subuinit
M-subunit
S-subunit
35
Realtive Intensity (a.u.)
M
75
M
(2.4)
R
(2.0)
S
(1.0)
50
25
0
0
100
200
distance migrated (mm)
A.
Lane 1 = cell lysate;
lane 2 = ammonium sulfate pellet;
lane 3 = pooled fractions following Q-sepharose chromatography;
lane 4 = final pool (pooled fractions following Heparin FF chromatography and overnight dialysis
against storage buffer)
B. Quantitation of lane 4 band intensities. The peaks labeled R, M and S, correspond to the R-, Mand S-subunits respectively. The numbers in parentheses indicate the relative area under each
peak.
Assays for activity
Since these are DNA binding proteins that we are
attempting to isolate, all activities should be DNAdependent
And the proteins should bind DNA (or RNA)
DNA binding assays using gels, filter binding or
fluorescence based assays
ATPase assays
These assays provide information both on the
presence of activity, DNA-dependence of activity
and also provide the fraction of active protein in an
assay
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Absorbance at 340nm (a.u.)
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DNA-dependent
and
site specific ATPase activity of EcoR124I
2.0
no DNA
M13 RF
Phase I; 34 μ M/min
1.5
1.0
Phase II; 2.4 μ M/min
scDNA
0.5
23 μ M/min
0.0
0
linear
10
20
30
40
Time (min)
Stoichiometry for EcoR124I
60
125
1μM SSB
100
50
Rate (μM/min)
Fractional fluorescence (%)
SSB site size
2μM SSB
75
50
25
0
0
8μM nts
20
M13 ssDNA μM nts
30
12.4 nM/2nM DNA
= 6.2nM or 16% active
20
10
14 μM nts
10
40
30
0
0
10
20
[protein] (nM)
30
Sources of contamination
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Nucleases Nucleases Nucleases!!!
E. coli has 12 exonucleases, several endonucleases, phosphatases, ribonucleases
It is imperative that a variety of nucleases asays be done following purification to determine
whether any nucleases are present
These assays typically involve incubating the purified protein with radioactively labeled DNA
Subject the samples to electrophoresis and compare to control untreated DNA
Types of DNA used (in separate assays):
– Linear dsDNA
– ssDNA (M13 for example)
– Linear ssDNA (either oligonucleotides or linearized M13 mp8 ssDNA)
If nucleases with distinct polarities are anticipated, both 5’- and 3’-end labeled substrates should
be used
Nucleic acid – either DNA or RNA
– can be detected using spectrophotometric methods and via radioactive labeling