1
Protein expression and purification
IX. Protein quality
Radka Dopitová
Kód předmětu: C8980
Quality parameters of produced protein
purity/homogeniety
folding state
3D structure determination
Detail function analysis
High quality of produced protein
stability
Source of protein Extraction Separation Quality evaluation
Recombinant protein quality evaluation
MIPFE (Minimal Information for Protein Functional Evaluation)
We consider that information concerning
aggregation status and secondary
structure should be reported as a minimal
requirement for publication under
Supplementary Material. These controls
should be available when authors describe
protein production as well as protein
interaction experiments (pull-down,surface
plasmon resonance, antibody/protein
microarrays, and isothermal titration
calorimetry).
Structural Proteomics In Europe (SPINE) consortium
- 3 year project, over 20 laboratories
- investigation of the effect of the biophysical properties of purified proteins
on their crystallizability
Geerlof et al., 2006
Quality assessment of protein preparation
The strategy is based on the experiences within SPINE
and the results of quality assessment survey.
Scheme for quality assessment of purified protein
Important biophysical properties for quality assessment of a protein preparation
and the most frequently used methods for their determination.
Biophysical property Methods used for its determination
Purity, homogeniety SDS-PAGE, NATIVE PAGE, size-exclusion chromatography,
mass spectrometry
Monodispersity, aggregation state Dynamic light scattering, size–exclusion chromatography,
analytical ultracentrifugation
Conformational state Static light scattering, analytical ultracentrifugation, size–
exclusion chromatography
Folding state Nuclear magnetic resonance spectroscopy, circular
dichroism, Fluorescence emission spectroscopy, Fouriertransformed
infrared spectroscopy
Stability Differential scanning calorimetry, circular dichroism, thermal
shift assay
Identity, modification Mass spectrometry
PROTEIN PURITY/HOMOGENIETY
Purity = protein is without contaminations by other proteins
Homogeniety/heterogeniety
– heterogeniety in pure macromolecule = MICROHETEROGENIETY of the sample
POSSIBLE SOURCE OF MICROHETEROGENIETY in protein sample:
- variation in primary structure (genetic variation, degradation)
- variation in folding (errors in folding or partial unfolding, conformers)
- variation in quarternary structure (oligomerization)
- variation in posttranslation modifications
- various aggregate states
- partial oxidation (e.g. sulfuhydryl groups in proteins)
- aging (e.g. deamination in proteins)
How to determine protein purity/(micro)homogeniety?
Ø polyacrylamide gel electrophoresis (SDS, native)
Ø size- exclusion chromatography
Ø mass spectrometry (MS)
Ø dynamic light scattering
Ø analytical centrifugation
PROTEIN PURITY/HOMOGENIETY
PROTEIN PURITY
SDS-PAGE
Ø For a pure protein, only one band is expected (20 mg of protein/lane of the gel).
Ø Mw determination (information about protein subunits)
Ø Protein purity is evaluated in SDS-PAGE using various software (e.g. Quantity
One from Bio-RAD).
Ø Definition of protein purity: a quantity of particular band as measured by its
intensity, expressed as a percentage of the total intensity of all bands in the lane of
the gel.
28% purity 80% purity 95% purity
Ø NATIVE PAGE
Ø PFO NATIVE PAGE
Ø perfluoro-octanoic acid (PFO) is a non-dissociative detergent that preserves
interactions within protein oligomers and thus permits molecular mass determination of
multimeric proteins.
AHP3 AHP5
0 0.05 1 0 0.05 1 % Tween20
AHP3
AHP5
15% SDS PAGE
10% NATIVE PAGE
15 % PFO PAGE10 % NATIVE PAGE
AHP2
PROTEIN HOMOGENIETY
Mass spectrometry (MS)
Ø Technique separates molecules according to their mass and charge.
Ø Mass spectrometer is composed of three parts: ionization source, an analyser, and a detector. The mass
analyzer separates protein ions according to their mass (m) to charge (z) ratios (m/z). Protein ions are
generated within the ionization source; the mostly common ionization technique are electrospray ionization
and matrix-assisted laser desorption ionization (MALDI).
Analysis of intact proteins: complete sequence coverage,
assessment of overall protein heterogeniety, identification of
labile PTMs.
Analysis of protein digest mixture: location of PTMs, amino acid
degradation products, glycosylation sites, and disulfide linkages
with specific regions of protein sequence.
Mass measurement accuracy ranging from 0.02% (ESI) to 0.1%
(MALDI) is routinely achieved.
Mass spectrometry (MS) – intact analysis
• Measurement of total mass of purified protein
Ø Applications: Evaluation of recombinant protein heterogeniety resulting from
posttranslation modifications, sequence variation generated from proteolysis, or
tranctriptional/translation errors, and degradation products, which are formed during
processing and final product storage.
40687
41887
0
100
200
300
400
Intens.[a.u.]
36000 38000 40000 42000 44000 46000 48000
m/z
glycoform
Non-modified protein
Analysis of posttranslation modification
23178
11584
3458
5181
0
1000
2000
3000
4000
5000
Intens.[a.u.]
3777
6951
8214 11572
4602
23183
2818
0
1
2
3
4
4
x10
Intens.[a.u.]
5000 10000 15000 20000 25000 30000
m/z
AHP2 protein - new sample
AHP2 protein - old sample
Mass spectrometry (MS)-intact analysis
Protein degradation Intact molecular mass
Size-exclusion chromatography (Gel filtration)
Superdex75 166023 01 07001:10_UV Superdex75 166023 01 07001:10_Fractions Superdex75 166023 01 07001:10_Inject
Superdex75 166023 01 07001:10_UV@01,BASEM Superdex75 166023 01 07001:10_Logbook
0
100
200
300
400
500
600
mAU
45.0 50.0 55.0 60.0 65.0 70.0 75.0 80.0 85.0 ml
A3 A4 A5 A6 A7 A8 A9 A10 A11 A12 A13 A14 A15 B15 B14 B13 B12 B11 B10 B9 B8 B7 B6 B5 B4 B3 B2 B1 C1 C2
46.42 51.00 57.36
65.01
27 03 2007 Superdex75 1660Valve7001:10_UV 27 03 2007 Superdex75 1660Valve7001:10_Conc
27 03 2007 Superdex75 1660Valve7001:10_Fractions 27 03 2007 Superdex75 1660Valve7001:10_Inject
27 03 2007 Superdex75 1660Valve7001:10_Logbook 27 03 2007 Superdex75 1660Valve7001:10_P960_Flow
0
50
100
150
200
250
300
mAU
35.0 40.0 45.0 50.0 55.0 60.0 65.0 70.0 ml
A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A12 A13 A14 A15 B15 B14 B13 B12 B11 B10 B9 B8
Cki rd
23.5 kDa
AHP3
22.3 kDa
• Size-exclusion chromatography separates proteins on the basis of size.
• Molecules move through a bed of porous beads. Smaller molecules diffuse further into the pores
of the beads and therefore move through the beads more slowly, while larger molecules enter less
or not at all and thus move through the beads more quickly.
• Both molecular weight and three-dimensional shape contribute to the degree of retention.
Dynamic light scattering (DLS)
Applications:
• Analysis of samples containing very broad distribudions of species of widely different sizes (e.g.
native protein and various sizes of large aggregates).
• DLS is a method to determine hydrodynamic size, polydispersities and aggregation effects of protein samples.
• The laser is focused in the sample cell and the particles scatter the light in all directions.
• DLS measures time dependent fluctuation in the intensity of the scattered light which occurs because the
particles are undergoing random Brownian motion. Analyses of these intensity fluctuations enables the
determination of the distribution of diffusion coefficients of the particles, which are converted into a size
distribution using Stokes-Einstein equation.
• Larger particles produce a very strong scattering intensity signal (detection of very small amounts of higher
mass species).
Dynamic light scattering (DLS)
Correlation function (average from 10 measurements) and
calculated hydrodynamic radius for a lysozyme sample. Results: Rh
= 1,95 nm, Polydispersity = 19,1%.
No protein aggregates are present. Such a DLS result indicates a
good chance for crystal growth, but it is not a guarantuee.
Monodisperse
sample
Polydisperse
sample
Results: A small peak is observed with a hydrodynamic radius of
6,21 nm (polydispersity 28,9%). Further broad peaks are visible at
41,4 nm and 212,5 nm and some dust particle in the micrometer
range.
The chances to get crystals from this protein sample are small. For
this sample a filtration step would be necessary to remove the large
particles that interfere with the smaller ones.
Otázka č. 1:
Vyjmenujte 3 metody pro stanovení mikrohomogenity/mikroheterogenity
proteinového vzorku.
Folding
Methods to study secondary, tertiary, and quaternary structure
Circular dichroism
Fluorescence emission spectroscopy
Size exclusion
chromatography
(gel filtration)
Conformational state Static light scattering, analytical ultracentrifugation, size–
exclusion chromatography
Folding state Nuclear magnetic resonance spectroscopy, circular
dichroism, Fluorescence emission spectroscopy, Fouriertransformed
infrared spectroscopy
• Amino acids with intrinsic fluorescence properties: phenylalanine, tyrosine, tryptophan; but only
tyrosine and tryptophan are used experimentally because their quantum yields (emitted
photons/excited photons) are high enough to give a good fluorescence signal. So this technique is
limited to proteins having either Trp or Tyr, or both.
• At an excitation wavelength of 280 nm, both Trp and Tyr will become excited. To selectively excite
Trp only, a 295 nm wavelength must be used.
• Trp and Tyr residues can be used to follow protein folding because their fluorescence properties
(quantum yields) are sensitive to environment, which changes when a protein folds/unfolds. In the
native folded state, Trp and Tyr are generally located within the core of the protein, whereas in a
partially folded or unfolded state they become exposed to solvent.
1. In a hydrophobic environment (buried within the
core of the protein), Tyr and Trp have a high
quantum yield and therefore a high fluorescence
intensity. In a hydrophilic environment (exposed to
solvent), on the other hand, their quantum yields
decrease, leading to low fluorescence intensity.
2. The wavelength of the emission maximum also
reflects the hydrophobic (l em 308 nm for azurin
indicating that Trp is deeply buried within the core
of the protein) and hydrophilic (l em 352 nm for
glucagon indicating that Trp is exposed to the
solvent) environment.
Fluorescence emission spectroscopy
Two modes of measurement
Ø As mentioned, the previous technique is limited to proteins containing Trp or Tyr residues. It is also
possible to use probes that bind specifically to hydrophobic protein residues (Sypro Orange, Nie red,
dapoxyl sulfonic acid, bis-ANS, 1,8-ANS ). Those surfaces are hidden in a native protein but exposed
in partially or fully unfolded proteins.
Measurement of fluorescence intensity
The thermal shift assay result for the screen of
reducing agents for AHP2 protein.
More in protein stability part….
NATIVE FLUORESCENCE
X
Fluorescence of probe – Sypro Orange
T (°C)
Circular dichroism spectroscopy
The technique measures the difference in absorbance between left and right
circularly polarized light beams, which arise due to structural asymetry.
The relevant spectral region is in far UV, i.e. from 240 nm down to 180 nm, where
peptide bonds absorb light. In this region, the different types of regular secondary
structure, such as alpha-helix and beta-sheet, exhibit a characteristic spectral
pattern.
- CD spectroscopy are used to assess the proper folding and characterize
secondary (and tertiary) structure of proteins
a-helix spectrum shows the characteristic two negative minima at 208 and 222 nm
and a large-magnitude positive maximum at 193 nm.
b-sheet spectrum shows a single negative minimum at about 215 nm and a positive
maximum at 196 nm (both of these are much smaller than the signal for a-helix).
The random coil (or disordered protein) shows only a small negative minimum at
about 198 nm.
The absence of regular structure results in zero CD intensity.
Circular dichroism spectroscopy – exploring the secondary structure of proteins
a-helix
b-sheet
random coil
Circular dichroism spectroscopy – applications
2. Comparing CD spectras of different mutants
of the same protein (maize b-glucosidase and its
mutants, structure possesses (b/a) 8 barrel).
1. Comparison of AHP2 protein purified either under
reducing or non reducing conditions. (AHP2Arabidopsis
thaliana histidine phosphotransfer
protein 2, structure possesses bundle of 6 a-
helices).
-15
-10
-5
0
5
10
15
20
25
30
185 200 215 230 245 260
wavelength [nm]
meanresidueellipticity
[x103
deg.cm2
.dmol-1
]
wt
F193A
F200K
W373K
F461L
P2
P3
P4
3.
3. Studying the secondary structure under
different conditions. Far-UV spectra of CGRP
(calcitonin gene related peptide) in
phosphate buffer of pH 7 (dashed line) and in
the same buffer + 50% trifluoroethanol (solid
line).
-40000
-30000
-20000
-10000
0
10000
20000
30000
40000
50000
60000
70000
180 190 200 210 220 230 240 250 260 270
wavelength (nm)
meanresidueellipticity(x103deg.cm2.dmol-1)
AHP2_1
AHP2_3
AHP2-5
AHP2 purified by AEC
under reduction
condition
AHP2 purified
by AEC without
reduction agent
Determination of quaternary structure – size exclusion chromatography (SEC)
• Size exclusion chromatography may be used to analyze the molecular size of macromolecules
after column calibration.
• Both molecular weight and three-dimensional shape of proteins contribute to the degree of their
retention.
Enzyme Apparent molecular
weight (kDa)
Dimer Monomer
WT 107 41
E401D 114 42
F193A 117 43
F200K 109 46
W373K 109 44
F461L 109 40
SEC
Native PAGE
Theoretical Mw:
Dimer/monomer 120 kDa/ 60 kDa
maize b-glucosidase and its mutant forms
CB
AS
• A technique to provide a direct measure of average molecular mass of the sample using the
relationship between the time-average intensity of light scattered by a molecule and its molecular
weight and size, as described by the Rayleigh theory. (In the simplest terms, Rayleigh theory says
that larger molecules scatter more light than smaller molecules from a given light source and that
the intensity of the scattered light is proportional to the molecule’s molecular weight).
„Classical“ (also "static" or "Rayleigh" ) light scattering
Applications:
• Determining whether the native state of a protein is a monomer or a higher oligomer.
• Measuring the masses of aggregates or other non-native species.
• The accurate determination of molecular weight from SLS data is absolutely dependent on the
purity of the protein sample.
http://www.ap-lab.com/light_scattering.htm
Static light scattering (SLS) in combination with size-exclusion chromatography (SEC)
• SLS in non-invasive method
• mass determination accuracy: 2-5%
• SEC elution profile shows two distinct species of the
protein.
• Comparison of elution peaks with standard curve
results in estimated Mws of proteins that do not
correspond well to defined oligomeric states.
• SLS clearly demonstrate that the two peaks
correspond well to monomeric (7.3 kDa) and dimeric
(13.7 kDa) forms of the protein.
• SLS measures the average Mw of the sample, the use of SLS data allows selection of elution
fractions that contains only single species, rather than mixture of monomer and dimer.
• The SEC-MALS detector measures the light scattered by a sample at many angles.
(Geerlof et al., 2006)
Concentration
detectors
Molecular mass of each
protein fraction coming
off the column
Otázka č. 2:
Přiřaďte jednotlivé metody k typu proteinové struktury, kterou
lze jimi analyzovat.
Sekundární
Terciární
Kvartérní
Gelová filtrace (GF)
Fluorescenční emisní spektroskopie (FES)
Cirkulární dichroismus (CD)
Protein Stability
• Protein stability is the net balance of forces, which determine whether a
protein will be in its native folded conformation or a denatured state.
• Protein stability normally refers to the physical (thermodynamic) stability,
not the chemical stability (loss of integrity due to bond cleavage, oxidation
of several residues etc.).
• Physical (thermodynamic) instabilities include aggregation and
precipitation, adsorption to surface, and protein unfolding.
Principle of thermodynamic stability
Pfolded P unfolded
A
• The net stability of a protein is defined as the difference in free energy between the
native and denatured state.
• The energy difference between these two states in physiological conditions is quite
small, about 5-15 kcal/mol.
DG
F
U
The midpoint temperature Tm of the unfolding transition is
defined as the temperature at which concentration of native
folded and unfolded protein is equivalent: [Pfolded] = [Punfolded].
Then the equilibrium is
K = [Pfolded] / [Punfolded] =1
Thus the free energy: DG = 0 (DG = - RTlnK)
DG is temperature – dependent
folded
unfolded
Tm
Protein stability profiling assays monitor the amount of
unfolded protein in solution as function of temperature.
Methods used to test protein thermodynamic stability:
• Differential scanning calorimetry measures the enthalpy (∆H) of protein
unfolding due to heat denaturation.
• Circular dichroism monitors specific changes of protein structure with
increasing temperature at single wavelength (220 nm).
• Differential static light scattering monitors the denaturation and subsequent
aggregation of unfolded proteins.
• Differential scanning fluorimetry (Tm shift assay, protein stability shift assay,
thermal shift or thermofluor) uses native fluorescence or an environmentally
sensitive dye to monitor protein unfolding.
Tm TmTm˂ ˂
• Protein will be most stable at the conditions where Tm
is the highest.
• DTm larger than 2°C is considered as significant.
• A positive DTm can be coupled to an increase in
structural order and a reduced conformational flexibility.
• A negative DTm indicates that buffer induces protein
structural changes towards a more disordered
conformation or it can be a sign of misfolding.
25 mM PIPES pH 6.5, 100 mM NaCl
25 mM PIPES pH 7, 100 mM NaCl
25 mM PIPES pH 7, 500 mM NaCl
Methods used to test protein thermodynamic stability
Protein stability in solution is influenced by: • temperature
• pH
• buffer type
• salt type and its concentration
• metal ions
• surfactants
• ligands….
High-throughput method is necessary to analyze the conditions which stabilize/destabilize the protein.
Monitoring solution-dependent changes in protein stability
Thermal shift assay
Instrumentation:
• Fluorescent Plate reader with heated stage
• Real-time PCR machine
Reagents:
• Enviromentally sensitive fluorescence dye, such as
Sypro Orange
Principle:
• Sypro Orange fluorescence is quenched in an
aqueous environment.
• As the temperature rises, the protein undergoes
thermal unfolding and exposes its hydrophobic core
regions.
• Sypro Orange then binds to the hydrophobic regions
and becomes unquenched.
• Fluorescence is monitored and plotted versus
temperature.
• The midpoint of the protein unfolding transition is
defined as the Tm.
• A significant difference in the shape of the curves could be observed between the proteins
that have crystallized (e.g., AC04, AC07, AD28) and the proteins that have not crystallized
(e.g., AC08, AD01, AD17).
• All of the proteins in our small sample set that have crystallized had a clear melting transition
combined with a low initial fluorescence intensity, whereas the melting curves for several of the
proteins that have not crystallized started at a high-fluorescence intensity and were lacking a
clear transition. The high initial fluorescence intensity is likely to be indicative of a high
hydrophobicity of the whole protein sample or a fraction of the protein sample.
Ericsson et al. 2006
Thermal shift assay result for the initial screen of different proteins
- information about the condition of proteins
Thermal shift assay results
Ericsson et al. 2006
Melting temperatures of the AC07 protein in the presence of different buffers
Mezzasalma et al., 2007
Protein purification and aggregation before and after stability profiling
–optimized conditions
Original purification conditions:
Ni NTA Superflow resin
Lysis/column wash buffer: 25 mM HEPES pH 7.5,
400 mM NaCl, 10% glycerol, 1 mM glutathione, 20
mM imidazole, 0.1 mM PMSF, 1x EDTA free
protease inhibitors
Altered purification conditions:
TALON metal affinity resin
Column wash buffer: 25 mM KH2PO4 pH
7.5, 5% glycerol, 5 mM imidazole.
Size-exclusion chromatography and SDS PAGE were used to gauge protein quality, purity and
tendency to aggregate.
Aggregation was the biggest challenge.
Aggregation was minimized.
cFMS
protein
Otázka č. 3.
Vyberte 3 křivky tání, které mají optimální tvar. Poté seřaďte proteiny, kterým tyto
křivky odpovídají podle vzrůstající thermodynamické stability (od nejnižší po
nejvyšší).
Thermal shift assay result for the initial screen of different proteins.
Chemical Stability
• Chemical instability refers to the formation or destruction of covalent bonds within
a polypeptide or protein molecule.
• These changes alter the primary structure of the protein and impact higher level of
its structure.
• Chemical instabilities such as deamidation and disulphide bond cleavage, may
also lead to physical instabilities, and vice versa.
• The common causes for chemical instability are
- deamination of asparagine and/or glutamine residues
– succiinimidation
– hydrolysis of the peptide bond of Asp residues at low pH
– oxidation
– proteolysis
– disulfide bond breakage and formation
– disulfide interchange at neutral pH
Succinimidation following deamination
• Formation of b-isoaspartate as a result of deamination of asparagyl residues or
isomerization of aspartyl residues is a major source of instability in proteins and
peptides, especially at neutral and alkaline pH after prolonged storage.
For instance, the disposition of an asparagine
residue followed by residue such as glycine,
serine or threonine in a polypeptide chain can
lead to deamination of an asparagine and
formation of a-aspartic acid and/or bisoaspartate
via an intramolecular
rearrangement that produce a succinimide
(cyclic imide, unstable under physiological
conditions) intermediate. This phenomenon
occurs at a rapid rate and conveys extra
negative charge to the protein.
1 : 3
The relative elution order of the iso-Asp, Asn, Asp, and succinimide forms of the 300VVSVLTVVHQDWLNGK315 tryptic
peptide from a recombinant human monoclonal antibody (Chelius, Rehder, &Bondarenko, 2005). The data demonstrate the
effect of incubation time on succinimide and iso-Asp/Asp formation forthe 300–315 tryptic peptide following
incubation in 100 mM Tris, pH 7.5 at 37°C for 4 hr, 18 hr, and 4 days. Note the approximately 3:1 ratio of iso-Asp to
Asp in the 18 hr and 4 day data.
(Srebalus Barner and Lim, 2006)
Analysis of succinimidation following deamination
LC/MS analysis
OTHER CHEMICAL REACTIONS AFFECT PROTEIN STABILITY
Hydrolysis
• Asp-Y bond may be at least 100 times more labile than other peptide bonds in
dilute acid. Cleavage is particularly rapid at Asp-Gly and Asp-Pro.
• Specific cleavage at Asp-Pro peptide bonds in a polypeptide chain can occur by
exposure to acid conditions (e.g. 10% acetic acid with pH 2.5, 70–75% formic acid)
at moderate temperatures (37°C, 40°C) for periods up to 120 hours.
Pyroglutamate formation
• Cyclization of amino terminal glutamine residues to pyroglutamyl residues
under mild acidic conditions is a frequent cause of protein modification.
Protein oxidation
• The side chain of cysteine, methionine, histidine, tyrosin and tryptophan residues are potential
sites of oxidation.
• Oxidation at these sites can be catalyzed by trace amount of transition metal ions (site-specific
process) or enhanced by oxidants or upon exposure to light (non-specific process).
• Oxidation can lead to protein aggregation, inactivation, increasing immunogenicity and encourage
proteolysis.
• The most easily oxidizable sites are on Met and Cys.
• Met residues in protein can be oxidized by atmospheric oxygen in vial containin only 0.4 %
oxygen. The rate of Met oxidation depend on the position in protein and pH.
http://www.ionsource.com/Card/MetOx/metox.htm
Amino Acid
Residue
Compositio
n
Residue
Monoisotopic Mass
Delta
Mass
Methionine C5H9NOS 131.0405 0
Methionine
Sulfoxide
C5H9NO2S 147.0354 15.9949
Methionine
Sulfone
C5H9NO3S 163.0303 31.9898
Sulfur S 31.9721 Detection
of oxidized Met species by MS
Mass spectrometry (MS)- Analysis of disulfide linkage
‘‘H’’ and ‘‘L’’ denote heavy chain and light chain of IgG, respectively.
Example: In a properly folded IgG antibody, all cysteins are involved in disulfide bonding and thus are
responsible for maintaining the characterisitic 3D structure of IgG antibody. Incomplete disulfide linkage or
free SH groups can lead to (a) the formation of antibody fragments that do not bind antigen and to (b)
antibody aggregation.
(B) Reduced peptide mixture:
1. Alkylation of free disulfide groups.
2. Digestion with protease
3. Reduction of disulfide bonds with
DTT
LS/MS analysis
(A)Non- reduced peptide
mixture:
1. Alkylation of free sulfhydryl
groups.
2. Digestion with protease
(Barnes and Lim, 2006)
- DTT
+ DTT
Protein oxidation- reducing agents
• Reducing agents that are primarily used to protect protein from oxidation (particularly of cysteine and methionine
residues): dithiothreitol (DTT) or 2-mercaptoethanol (2-ME), tris(2-carboxyethyl)phosphine (TCEP), EDTA (or
EGTA).
• The higher the pH and temperature, the shorter the half-lives of the thiol reagents.
• Metal chelators such as EDTA at final concentation of 1-5 mM avoid metal (Cu, Fe, and Zn) -induced oxidation of
SH groups and helps to maintain the protein in reduced state.
•TCEP is powerfull a useful reductant over a much wider pH range (1.5–8.5) than is DTT.
•For long-term storage of proteins, TCEP is significantly more stable than DTT without metal chelates such as
EGTA in the buffer, whereas DTT is more stable if metal chelates are present.
• The choice of reductant is application specific.
Half-life of thiol compounds in
solution (hours)
Conditions 2-mercaptoethanol DTT
pH 6.5, 20°C > 100 40
pH 7.5, 20°C 10 10
pH 8.5, 20°C 4 1.4
pH 8.5, 0°C 21 11
pH 8.5, 40°C 1 0.2
pH 8.5, 20°C + 0.1 mM Cu 2+
0.6 0.6
pH 8.5, 20°C + 1 mM EDTA >100 4
All thiols compounds
were dissolved in 0.1 M
potassium phosphate
buffer.
PROTEOLYSIS
Incubation of sample (extract or purified protein) at 30°C-37°C at various time points
Take aliquots at various time points and assay them for biological activity (Caution: as the target
protein may be partially degradated, yet retain full biological activity) or protein size and
microheterogeneity have to be monitored by SDS-PAGE and following western blotting and/or by
mass spectrometry.
If the proteolysis is a problem, then protease inhibitors must be added to the sample and during all
future attempts at purification of that target protein.
A simple test for proteolytic degradation
• Proteolysis decreases a protein size, make it less stable and modify its charge,
hydrophobicity, activity as a catalyst or immunogenic properties.
+EDTA - EDTA
Products of
degradation
PROTEOLYSIS
Protein was co-purified with traces of metalloproteases.
2 weeks,
4°C
PROTEIN CONCENTRATION AFFECTS STABILITY
Ø In general, proteins are less stable at low concentrations (<50 mg/ml). Under these
conditions, multiple subunit proteins and cofactors tend to dissociate.
Ø At the protein concentration falls below ~50 mg/ml, protein loss can occur due to strong and
irreversible adsorption of the protein to a variety of surfaces, including glass, plastic, and various
types of filtration media used for concentrating, clarifying, or sterilizing proteins.
Ø It is advisable to keep protein concentration as high as possible (e.g. >1 mg/ml) during
purification and storage. This is relatively easy to achieve during the early stages of a
purification procedure; however, it becomes more difficult during the later stages of purification
or in the case of purification of low abundant proteins isolated from natural sources.
Ø Protein solutions lower than 1 mg/ml should be concentrated as rapidly as possible using
methods such ultrafiltration or lyophilization or precipitation using TCA or (NH4)2 SO4 and after
dilution in small volume.
Ø When the rapid concentration of a dilute protein solution is not possible, the addition of ~1
mg/ml bovine serum albumin (BSA) or solvent additives, such as polyethylene glycol (0.05% v/v)
and nonionic detergents (0.02% w/v, Triton-X-100, or Tween-20) to all buffers and eluents used
in the purification procedure has been shown to be very effective.
Ø On the other hand, protein aggregation is generally concentration dependent.
Protein aggregation – a major event of physical and chemical instability of proteins
• Protein aggregation is the assembly of individual protein molecules into amorphous,
multimeric states, which results in many cases from intermolecular association of
partially denatured protein chains.
• Protein aggregation may be induced by a variety of physical factors, such as
temperature, ionic strength, foaming, protein concentration, pH shift, vortexing, etc.
• Protein aggregates may have reduced or no activity, solubility, and altered
immunogenicity.
• Hydrophobic interaction, i.e. the reluctance of non-polar groups to be exposed to
water, is considered to be a major driving force for both protein folding and
aggregation. Both protein aggregation and folding represent a balance of exposed
and buried hydrophobic surface areas. The balance is so delicate that a change of
one amino acid in a protein may substantially change its aggregation behavior.
Mechanisms of protein aggregation
Philo and Arakawa, 2009
Mechanism 1: Reversible association of the
native monomer
The tendency to reversible associate is intrinsic to
the native form of the protein. There may be
complementary patches on the monomer surface
to form reversible small oligomers. With
increasing concentration larger and larger
oligomers form, over time these larger aggregates
often become irreversible.
Mechanism 2: Aggregation of
conformationally- altered monomer
The native monomer transiently undergoes a
conformational change or partial unfolding.
Altered conformation of monomer associates
strongly. This aggregation mechanism appears to
be the dominant one for many protein.
Aggregation wil be promoted by stress such as
heat or shear.
Mechanisms of protein aggregation
Mechanism 3: Aggregation of chemically –
modified product
The change in protein conformation that precedes
aggregation is caused by differences in covalent
structure. This difference is usually caused by
chemical degradation such as oxidation of methionin,
deamination, or proteolysis. Chemical changes may
for example create a new sticky patch on the
surface, or change the electric charge in a way that
reduces electrostatic repulsion between monomers.
Mechanism 4: Nucleation – controlled
aggregation
Nucleation- controlled aggregation is a common
mechanism for formation of visible particulates or
precipitates. In this mechanism the native
monomer has a low tendency to form small or
moderately- sized oligomers. However if an
aggregate of sufficient size manages to form, then
the growth of “critical nucleus” through addition of
monomers is strongly favored and the formation of
much larger species is rapid. Philo and Arakawa, 2009
Mechanisms of protein aggregation
Mechanism 5: Surface- induced aggregation
starts with binding of the native monomer to a
surface. In the case of air/liquid interface that
binding would be driven by hydrophobic
interactions, but for a container favorable
electrostatic interactions might also be
involved. After this initial binding event the
monomer undergoes a change in conformation
and the it is the same as mechanism 2.
To avoid protein aggregation the experimental conditions have to be test very carefully
with regards to all type of aggregation mechanisms.
Philo and Arakawa, 2009