1
Protein expression and purification
IX. Protein quality
Lubomír Janda, Blanka Pekárová, Radka Dopitová and Lukáš Žídek
Kód předmětu: C8980
Recombinant protein quality evaluation
MIPFE (Minimal Information for Protein Functionally 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).
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
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.
Mass spectrometryIdentity, modification
Differential scanning calorimetry, circular dichroism, thermal
shift assay
Stability
Nuclear magnetic resonance spectroscopy, circular
dichroism, Fluorescence emission spectroscopy, Fouriertransformed
infrared spectroscopy
Folding state
Static light scattering, analytical ultracentrifugation, size–
exclusion chromatography
Conformational state
Dynamic light scattering, size–exclusion chromatography,
analytical ultracentrifugation
Monodispersity, aggregation state
SDS-PAGE, size-exclusion chromatography, mass
spectrometry
Purity
Methods used for its determinationBiophysical property
PROTEIN PURITY/HOMOGENIETY
Purity = protein is without contaminations by other proteins
Homogeniety/heterogeniety
– heterogeniety in pure macromolecules = 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)
- variation in quarternary structure (oligomerization)
- variation in posttranslation modifications
- various aggregate states
- oxidation (e.g. sulfuhydryl groups in proteins)
- aging (e.g. deamination in proteins)
How to determine protein purity/(micro)homogeniety?
electrophoresis (SDS, native)
size- exlusion chromatography
mass spectrometry (MS)
dynamic light scattering
analytical centrifugation
PROTEIN PURITY/HOMOGENIETY
PROTEIN PURITY
SDS-PAGE
For a pure protein, only one band is expected (20 µg of protein/lane of the gel).
Mw determination (information about protein subunits)
Protein purity is evaluated in SDS-PAGE using various software (e.g. Quantity
One from Bio-RAD).
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
NATIVE PAGE
PFO (pentadecafluorooctanic acid) NATIVE PAGE
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 PURITY/HOMOGENIETY
Mass spectrometry (MS)
Technique separates molecules according to their mass and charge.
Mass spectrometer are 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).
Application: 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.
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) was routinely achieved.
Mass spectrometry (MS) – intact analysis
• Measurement of total mass of purified protein
- verification of construct
- information about degradation products, contaminants, posttranslation modifications
40687
41887
0
100
200
300
400
Intens.[a.u.]
36000 38000 40000 42000 44000 46000 48000
m/z
glycoform
Non-modified protein
23178
11584
3458
5181
0
1000
2000
3000
4000
Intens.[a.u.]
5000 10000 15000 20000 25000 30000 35000
m/z
Posttranslation modificationPurity
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
New sample
Old sample
Mass spectrometry (MS)-intact analysis
- information about protein degradation
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 bed 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.
• Measurement of the velocity of the Brownian motion of molecules and/or particles.
• 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 fluctuation anables 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.
Folding
Methods to study secondary, tertiary, and quaternary structure
Circular dichroism
Fluorescence emission spectroscopy
Gel filtration
• 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 (λ em 308 nm for azurin
indicating that Trp is deeply buried within the core
of the protein) and hydrophilic (λ em 352 nm for
glucagon indicating that Trp is exposed to the
solvent) environment.
Fluorescence emission spectroscopy
Two modes of measurement
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 folded or fully unfolded proteins.
Measurement of fluorescence intensity
The thermal shift assay result for the initial screen of
23 different buffers with pH range 4.5 - 9.
More in protein stability part….
NATIVE FLUORESCENCE
X
Fluorescence of probe – Sypro Orange
Circular dichroism spectroscopy
The technique depends on 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 spectra are used to assess the proper folding and the secondary
structure of proteins
α-helix spectrum shows the characteristic two negative minima at 208 and 222 nm
and a large-magnitude positive maximum at 193 nm.
β−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 α-helix).
The random coil (or unordered) shows only a very small signal above 210 nm and 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
α-helix
β-sheet
random coil
Circular dichroism spectroscopy – applications
2. Comparing the structures of different mutants
of the same protein (maize β-glucosidase its
mutants, structure (β/α) 8 barrel).
1. Determining whether a protein is correctly folded.
(Arabidopsis thaliana histidine phosphotransfer
protein 2, structure bundle of 6 α-helices).
Comparison of AHP2 purified either under
reducing or non reducing conditions.
-15
-10
-5
0
5
10
15
20
25
30
185 200 215 230 245 260
wavelength [nm]
meanresidueellipticity
[x10
3
deg.cm
2
.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)
ΑΗΠ2_1
ΑΗΠ2_3
ΑΗΠ2−5
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.
40109F461L
44109W373K
46109F200K
43117F193A
42114E401D
41107WT
MonomerDimer
Apparent molecular
weight (kDa)
Enzyme
SEC
Native PAGE
Theoretical Mw:
Dimer/monomer 120 kDa/ 60 kDa
maize ββββ-glucosidase and its mutant forms
CB
AS
• Also known as "static" or "Rayleigh" scattering.
• Provides a direct measure of average molecular mass of the sample.
• Static Light Scattering a beam of polarized light is focused onto a polymer/particle and the
scattered light is detected with a photodiode detector.
• The intensity of this scattered light is measured, which is proportional to the molar mass and the
concentration of protein(s) in solution (ILS ~ Mw * c).
„Classical“ 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 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.
(Geerlof et al., 2006)
Concentration
detectors
Molecular mass of each
protein fraction coming
off the column
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.).
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.
∆G
F
U
The midpoint temperature Tm of the unfolding transition is
defined as the temperature at which concentration of native
and non-native protein is equivalent: [Pfolded] = [Punfolded].
Then the equilibrium is
K = [Pfolded] / [Punfolded] =1
Thus the free energy: ∆G = 0 (∆G = - RTlnK)
∆G 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 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 an environmentally sensitive dye to monitor
protein unfolding.
Tm TmTm
• Protein will be most stable at the conditions where Tm
is the highest.
• ∆Tm larger than 2°C is considered as significant.
• A positive ∆Tm can be coupled to an increase in
structural order and a reduced conformational flexibility.
• A negative ∆Tm 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 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).
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
Chemical Stability
• Chemical stability involves loss of integrity due to bond cleavage.
• Examples of some degradation reactions in proteins:
– 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 β-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 α-aspartic acid and/or βisoaspartate
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 h r, 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 u p 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 thio groups 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 -31.9721SSulfur
31.9898163.0303C5H9NO3S
Methionine
Sulfone
15.9949147.0354C5H9NO2S
Methionine
Sulfoxide
0131.0405C5H9NOSMethionine
Delta
Mass
Residue
Monoisotopic Mass
Residue
Compositio
n
Amino Acid
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)
Protein oxidation-reducing agents
• Reducing agents that are primarily used to protect free SH-groups 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.
• TCEP is a more stable, faster and stronger reductant than DTT at pH values below 8.0. It resists air oxidation
and is stable over a wide pH range (1.5–8.5).
• TCEP is recommended for long-term storage of proteins in the absence of metal chelators (EDTA, EGTA),
because Fe3+ and Ni2+ catalyzes DTT oxidation.
• 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.
4>100pH 8.5, 20°C + 1 mM EDTA
0.60.6pH 8.5, 20°C + 0.1 mM Cu 2+
0.21pH 8.5, 40°C
1121pH 8.5, 0°C
1.44pH 8.5, 20°C
1010pH 7.5, 20°C
40> 100pH 6.5, 20°C
DTT2-mercaptoethanolConditions
Half-life of thiol compounds in
solution (hours)
All thiols compounds were dissolved in 0.1 M potassium phosphate buffer.
PROTEOLYSIS
Incubatation of sample (extract or purified protein) at 30°C-37°C at various time points
Take aliquats 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
In general, proteins are less stable at low concentrations (<50 µµµµg/ml). Under these
conditions, multiple subunit proteins and cofactors tend to dissociate.
At the protein concentration falls below ~50 µµµµg/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.
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.
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.
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.
On the other hand, protein aggregation is generally concentration dependent.
Protein aggregation – a major event of physical 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.
• Proteins aggregate to minimize thermodynamically unfavorable interactions between a solvent
and exposed hydrophobic protein residues.
• 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.
• Other possible mechanisms: formation of non-native disulfide bonds, electrostatic interactions.
Mechanisms of protein aggregation
Philo and Arakawa, 2009
Mechanism 1: 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: 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.
This type of aggregation will be inhibited by
excipients or conditions that stabilize the native
conformation.
Mechanisms of protein aggregation
Mechanism 3: 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
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
Improving chemical stability will reduce aggregation
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