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Important Factors Influencing Protein Crystallization

Article · September 2016

DOI: 10.17352/gjbbs.000008

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 Global Journal of Biotechnology and Biomaterial Science

Mohnad Abdalla1*, Wafa Ali Eltayb1, Mini Review

Abdus Samad1, Elshareef SHM1 and
TIM Dafaalla2
1
Hefei National Laboratory for Physical Sciences at the
Important Factors Influencing
Microscale and School of Life Sciences, University of Science
and Technology of China, Hefei, Anhui 230027, People’s
Republic of China
Protein Crystallization
2
College of Plant Science, Jilin University, Changchun
130062, China Abstract
Dates: Received: 16 September, 2016; Accepted:
The solution of crystallization problem was introduced around twenty years ago, with the
29 September, 2016; Published: 30 September,
introduction of crystallization screening methods. Here reported some of the factors which affect
2016
protein crystallization, solubility, Concentration of precipitant, concentration of macromolecule, ionic
*Corresponding author: Mohnad Abdalla, strength, pH, temperature, and organism source of macromolecules, reducing or oxidizing environment,
Hefei National Laboratory for Physical Sciences additives, ligands, presence of substrates, inhibitors, coenzymes, metal ions and rate of equilibration.
at the Microscale and School of Life Sciences, The aim of this paper to give very helpful advice for crystallization.
University of Science and Technology of
China, Hefei, Anhui 230027, PR China, E-mail:
Buffer is most straight forward way to make a crystallization
www.peertechz.com problem by effect the protein behave. There are many rules and
Keywords: Proteins; Crystallization; Isoelectric point; different protein crystallization methods, some of it has been
pH; Solubility; Stability developed during the recent years. However, Protein crystallization
still represents a great challenge for bio crystallography. The
Introduction cumulation of crystallization information in crystallization databases
and in structural articles it allow us to design crystallization
X-ray crystallography has provided 3D structures of thousands experiments depending on the character of the protein. Because
of proteins. In spite of these advances, many factors continue to there is no ideal procedure for crystallization, our article discusses
be problem that can lead to unsuccessful proteins crystallization. the crystallization problem try to simplify the composition of the
We always know theoretical pI, molecular weight and amino-acid crystallization and give some solution advice.
composition, while pH and salt concentration are some of the
Protein solubility
variables that can be expected from other similar known structure.
Yet, a protein behavior depends very much on the environment it Protein solubility is a common complex interaction problem
is in. between the physiochemical nature of the proteins, rate of protein
synthesis, amino acid composition, the Protein concentration, type
Proteins are generally present in a biological sample as their of salt present in the buffer, the concentration of the salt used, ionic
native state. They are very often associated with other proteins strength, pH, temperature, osmotic pressure, cellular location of
and integrated into large complexes. In most cases they are not expression, and cellular tools or chaperones are all important in
soluble in their innate state after isolation as result of that must be protein solubility. Protein solubility may decrease in the presence of
denatured to help solubilization and Crystallization. Initial protein complexes with lipids, nucleic acids or other nonprotein.
crystal commonly need to screen conditions using little as possible to
It is important to select better option for tag in the target proteins.
improve the optimization methods for protein crystallization. Unfortunately, there is no consolidated system for rapid screening to
Crystallization is thermodynamically process. Once begin, will differentiate the best tag.
continue under kinetic control until super saturation is lost. The Addition L-Arg and L-Glu at 50 mM to the buffer can increase
crystallization process affected by physical conditions of the solution, solubility up to 8.7 times, because they can in preventing protein
solution solubility, the presence of impurities, nucleation, solution aggregation and precipitation as well as increase the long-term
saturation and degree of super saturation, crystal growth, including stability also it did not prevent specific protein-protein and protein-
solution composition, pH and temperature, and to date is not fully RNA interactions [1].
understood.
Normally when there is solubility issues, the first thing we need
Prediction of secondary structure, solubility, domain organization, to check it the buffer composition, varying pH and salt strength it can
stability, signal peptide, hydrophobicity and pH, can provide useful make a big difference to solubility. Then try solubilising agents, e.g
information for crystallization strategies and salt prediction tools NDSBs or detergents. Protein solubility can improved by selecting
it need to be develop. in general proteins has low molecular weight only a soluble domain for expression, and delete the hydrophobic
domain. Moreover there are solubility prediction tools in the internet
easier for crystallization than high molecular weight, single-domain
it can be useful.
easier for crystallization than multi-domain and an oligomeric state
multimer more likely to crystallize than a monomer high molecular In order to be sure target protein is soluble culture need to be
weight. lysate (lysozime 1mg/ml on ice 1hour, it should be under non

Citation: Abdalla M, Ali Eltayb W, Samad A, Elshareef SHM, Dafaalla TIM (2016) Important Factors Influencing Protein Crystallization. Glob J Biotechnol
Biomater Sci 2(1): 025-028.
025
Abdalla et al. (2016)

denaturation condition) and centrifugation at 35000 x g. then use an polymorphs as result of different temperature. Lowering the growth
aliquot of supernatant for SDS-PAGE. In case of protein is soluble inducing temperature, this lessening the rate of protein synthesis and
well found in the supernatant. usually more soluble protein is obtained. Because the temperature
can affects solubility is better to cool the Crystal solutions to prevent
Detergents
protein degradation.
Detergents are commonly used at concentrations of 1–4%. It
Optimum salt concentration
useful in solubilizing by disrupt hydrophobic interactions between
and within proteins. The detergent used for solubilization does not Different NaCl concentrations with different pH levels it was
requisite to be the same for purification and crystallization, also use for crystallization. The effective of salt concentration in a
the presence of various detergents perhaps inhibit the activity of crystallization solution can be prognosticate before performing in the
different proteases. When despite all efforts not work then it needs experiment, because it is depended on the buffer pH and the pI of the
denaturation and solubilization of the target protein. This is done by protein. It is important for successful crystallization to keep the salt
a denaturing agent e.g. guanidine or urea under reducing conditions concentrations neither too low nor too high in the protein solution,
(~ 20 mM DTT). A western blot necessary for the supernatant before highly salt concentration may stabilize the protein buffer or decrease
and after the high-speed centrifugation will give an indication of how the protein solubility lead to precipitation. Then crystallization
much detergent was effect in the supernatant (membrane). happens in the drop in which the components are concentrated
through water loss. If the original proteins as well as reservoir solution
Reducing agents
do not contain enough salt, the concentration in the drop does not
Reducing agents are generally used in sample preparation to cleave reach the marginal concentration level.
disulfide bond DTT, DTE and β-ME are more commonly reducing
Commonly, the increase of precipitant concentration in crystal
agents used. The aim of using these agents to protect proteins from
solutions is an effective technique adopted to lowering the solubility of
precipitating and aggregating due to oxidative crosslinking and to
proteins. The salt it can stabilizes the water structure (Kosomtropic) or
reduce protein aggregation that may inhibit crystallization, Reducing
disrupts the water structure (Chaotropic). In addition, the solubility
agents can interact with metals within protein sample and that is not
of the proteins drop at high concentrations of salt as well as solubility
good for crystallization in this case. BME is particularly sensitive to
of the proteins increases at low concentrations of salt. The optimal
cobalt, copper and many phosphate buffers while DTT is sensitive to
salt concentration will slowly dehydrate our drop as well as increase
nickel. But until now no specific article show different between the
the concentration of our protein slowly.
DTT, DTE and β-ME as well as in which crystallization step is better
to be use. The marginal ionic strength become high when the difference
between the pI of the proteins and the pH of the buffer is large, which
Isoelectric point (pI), pH & Temperature
is agreement with the idea of the electrostatic screen effect of a salt [3].
The pH influence on protein solubility, different proteins
Multi subunit proteins or virus it is large macromolecules,
are soluble at different pH values, at high pH protein soluble
highly salt concentrations are not effective to promote enough
(deprotonate), low pH protein soluble (Protonated) and at isoelectric
interactions to induce crystallization while the Polyethylene glycol
point: protein aggregates. Acidic proteins has pI lower than 7, more
(PEG) is a commonly used as precipitant in protein crystallization is
likely to crystallize around one pH unit above their pI, while basic
indispensable to induce crystallization.
proteins more likely to crystallize lower than their pI about 1.5–3 pH
units [2]. Generally, protein aggregation or precipitation when the Crystallization of iron protein under anaerobic condition may
solubility decrease at pH close to the isoelectric point (pI). It better to facing precipitation problem, if the oxidation of the protein causing
move pH away from the pI to increase protein solubility and Super the precipitation try to add sodium dithionite to preserve the reduced
saturation. At low salt concentration the electrostatic repulsions well state, also is better to change the ratio in the crystallization drop by
decrease when pH is different from pI, this well likely induce PEG to higher the amount of the precipitant.
effect depletion attraction. The depletion attraction due to polymer is
Expression system
more effective when the protein net charge is lower.
E coli is first choice for structural biologists to produce protein for
To screen favor attraction between macromolecules and protein
X-ray crystallography, but sometime unsuitable to express membrane
charges it better to increasing ionic strength or gently moving pH
proteins in bacterial expression systems because the system cannot
closer to pI. The most critical things is pI we need to make Sure the
provide the post-translational modifications, the folding machinery
protein having net charge close to zero and it soluble, and this depend
and specific lipid environment. For eukaryotic membrane protein
on some paper advice.
better to express in Saccharomyces cerevisiae, Pichia pastoris, Sf9
Actually, temperature is important parameter for crystallization, insect cells [4] and human embryonic kidney.
it is better be screened. Temperature influences crystal growth and
Improve the expression level
nucleation by changing super saturation as well as solubility of the
sample. For instance, pH of Tris buffer is susceptible to temperature In case of poor expression it need to be sure the protein is
changes. However, temperature can useful at low ionic strengths not toxic for the cell. Choosing a smaller fragment of the target
by effects the amplified. Proteins very often show several crystal protein can improve the expression levels and solubility, because

Citation: Abdalla M, Ali Eltayb W, Samad A, Elshareef SHM, Dafaalla TIM (2016) Important Factors Influencing Protein Crystallization. Glob J Biotechnol
Biomater Sci 2(1): 025-028.
026
Abdalla et al. (2016)

E. coli may not express well very large proteins > 70 kDa. Using in Beta sheets, misfolding or mutation might result in exposure of
strains carrying mutations which remove the production of proteases hydrophobic residues. In this case, proteins aggregate to inhibit
can occasionally enhance accumulation by diminution proteolytic hydrophobic exposure as well as avoid entropic penalty. Beta sheets
degradation, also reducing the growth temperature will produce are stabilized through hydrogen bonding as well as hydrophobic
protein slow but at the same time slower proteolytic degradation. contacts, while Alpha helices are predominantly stabilized through
Using special media containing trace metals (high density culture hydrogen bonding. Thus sheet is a little inferior in terms of stability.
media) it can increase protein yield 10 time over LB. Addition of co-factors or prostethic groups (such as a vitamin,
Co-expression of foldases proteins e.g. peptidyl prolyl cis/ lipid, or inorganic like a metal ion) which are essential for protein
trans isomerases (PPI’s), disulfide oxidoreductase (DsbA), disulfide stability or for proper folding, but must be not fluctuation the pH
in the medium during growth, this leads to the accumulation of the
isomerase (DsbC) and protein disulfide isomerase (PDI) with the
target protein osmoprotectants in the cell, which stabilize the protein
target protein may lead to highly levels of protein solubility. The other
structure.
advantages for this enzymes assist the formation of disulfide bonds
which does not happen in the reducing environment an also reduced Biophysical methods like CD spectroscopy and other differential
proteolysis scanning fluorometry (DSF) can be used for characterizing the
stability of the protein in different buffers, pH and in the presence
Commonly challenging and important task, protein aggregation of different ligands to make sure that the protein is correctly folded.
and precipitation is often happen when increasing a protein There is several fold recognition websites (FOLDpro, RF-Fold,
concentration in the buffer to the required level. SSHMM, THREADER, BLASTLINK, SSEARCH, PSI-BLAST and
Size exclusion chromatography HMMER) used to predict the protein fold.

Is useful for further purification but some time is better to use Mutation
new column for purification. The shape of the chromatogram and When the native protein fails to crystallize against ~ 300500
retention time provides. crystallization conditions it was propose genetically modified
proteins whether by truncations or point mutations might better for
High homogeneity and purity of the sample are crucial for the
crystallization.
crystallization to be successful, the presence of different aggregates or
oligomeric forms in the protein solution it well effect, we need to use Mutants of Tyr →Phe and Thr→Val more stable than the wild-type
Dynamic light scattering (DLS) to check that or small-angle X-ray protein, but have little effect on the conformation of the protein [5].
scattering (SAXS). However, it has been reported that point mutations spectacular affect
the amount of aggregate formation in number of protein systems;
Folding and stability these include the interferon-γ, P22 tailspike protein, colicin A, single-
‘Natively unfolded’ proteins are some proteins unfolded in any chain antibodies, immunoglobulin domains, and interleukin-1β [6].
However, it is still not clear what mutations are likely to be helpful in
physiological conditions, and this kind may better to co crystallize
crystallization (Figure 1).
with other protein. UN folding, while highly beta strand are more
likely to form amyloid-like aggregates, but both of them can be Cysteine and histidine predominantly react with reagents
crystallize PDB (1aos--1by3). There is no direct correlation between prepared for the other and for other amino acid side chains as well,
percentage of helix secondary structure as well as beta strand and that why it is problematic during crystallization. Some domain and
stability. However, proteins with high alpha-helical protein habitually cloning are affecting the solubility this checking before expend time
are less problematic upon. There are more hydrophobic contacts on buffer optimization.

Figure 1: superdex 75 A. New superdex 75 B. Old superdex 75 as we can see new superdex 75 separate the protein to three pick, one is aggregation protein, two
is dimer protein, three is monomer, while the old superdex 75 cannot separate them and in this case is difficult to be crystal because the crystal condition for dimer
is not same to the monomer condition rather then that aggregation it can be crystal.

Citation: Abdalla M, Ali Eltayb W, Samad A, Elshareef SHM, Dafaalla TIM (2016) Important Factors Influencing Protein Crystallization. Glob J Biotechnol
Biomater Sci 2(1): 025-028.
027
Abdalla et al. (2016)

Nucleation bonds primarily to the pyrimidine or purine rings [9]. Tris decrease
the metal ion–catalyzed oxidation of Cys, consequently, Cys being
Sometime one protein can create different crystal, but that not
available for interaction with the other reagent.
mean all of them they are stable, it can just be kinetically favored.
There is two kind of nucleation: heterogeneous is the most common, Conclusion
occurs by interface of different composition and homogeneous as high
In this review presents important factors that affect protein
purity crystal. The nucleation rates it can be increased by increasing
crystallization and ways to improve the results, but more needs to be
the super saturation, solubility, viscosity and decreases temperature
done to better understand. Some proteins have ability to make crystal
and solid–liquid interfacial tension.
within initial concentration less than 1 mg per ml. Right working
Ethanol sequence and buffer well make all the difference.
In ethanol, the assist of peptide group burial to protein stability Acknowledgements
would be increase and the contribution of non-polar group burial
This work was supported by Ministry of Science and Technology
would be decrease. Because α-helices are expected to be stable
of China and the CAS-TWAS President’s PhD Fellowship. All authors
structures in ethanol, ethanol used to increase α-helix formation in
declare no conflict of interests.
proteins and peptides [7], also ethanol and methanol can lowers the
thermal denaturation temperature. References
Glycerol 1. Golovanov AP, Hautbergue GM, Wilson SA, Lian LY (2004) A simple method
for improving protein solubility and long-term stability. J Am Chem Soc 126:
Often use in crystallization to protect the proteins as cryosolvent 8933-8939.
because antifreeze properties and to enhance their solubility by 2. Kirkwood J, Hargreaves D, O’Keefe S, Wilson J (2015) Analysis of
stabilizing the conformation, sugar protein specially affected by crystallization data in the Protein Data Bank. Acta Crystallogr F Struct Biol
glycerol because of ribonuleotides bonds. But some time glycerol Commun 71: 1228-1234.

work as an antinucleation agent in crystallization. 3. Yamanaka M, Inaka K, Furubayashi N, Matsushima M, Takahashi S, et

al. (2011) Optimization of salt concentration in PEG-based crystallization
TRIS & HEPES buffers solutions. J Synchrotron Radiat 18: 84-87.

The differences in the influence of the buffers could be attributed 4. Jasti J, Furukawa H, Gonzales EB, Gouaux E (2007) Structure of acid-
sensing ion channel 1 at 1.9 A resolution and low pH. Nature 449: 316-323.
neither to disparity in the amounts of de-protonated buffer ions
nor to disparity in ionic strength. Tris and HEPES being positively 5. Pace CN, Trevino S, Prabhakaran E, Scholtz JM (2004) Protein structure,
charged at pH 7.0. Protein activity increased in different buffer by stability and solubility in water and other solvents. Philos Trans R Soc Lond B
Biol Sci 359: 1225-1234.
following order: citrate < Tris ≈potassium phosphate <HEPES. While
oligonucleotide initiator decreased in the following order: cacodylate 6. Izard J, Parker MW, Chartier M, Duche D, Baty D (1994) A single amino
acid substitution can restore the solubility of aggregated colicin A mutants in
> MES > HEPES > TRIS > phosphate [8]. Tris as well as HEPES Escherichia coli. Protein engineering 7: 1495-1500.
prevent the auto-oxidation. HEPES accelerated the degeneration rate
7. Luo P, Baldwin RL (1997) Mechanism of helix induction by trifluoroethanol:
of the oxidant and peroxynitrite. The exist of as much as 100 mM
a framework for extrapolating the helix-forming properties of peptides from
NaCl enhance the reversibility and stability of unfolding transitions trifluoroethanol/water mixtures back to water. Biochemistry 36: 8413-8421.
in Hepes buffer. The crystal structure obtain from HEPES buffer was
8. Arzumanov AA, Victorova LS, Jasko MV, Yesipov DS, Krayevsky AA
more similar to the active conformation. (2000) Terminal deoxynucleotidyl transferase catalyzes the reaction of DNA
phosphorylation. Nucleic acids research 28: 1276-1281.
Proteins in L-arginine and Tris buffer at pH 7.4 stay stable against
aggregation for longer periods of time. Tris is Good’s buffers bind to 9. Righetti PG, Gelfi C, D’Acunto MR (2002) Recent progress in DNA analysis
by capillary electrophoresis. Electrophoresis 23: 1361-1374.
the DNA not only by electro static interactions but also by hydrogen

Copyright: © 2016 Abdalla M, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Citation: Abdalla M, Ali Eltayb W, Samad A, Elshareef SHM, Dafaalla TIM (2016) Important Factors Influencing Protein Crystallization. Glob J Biotechnol
Biomater Sci 2(1): 025-028.
028
Affects of imidazole on protein solubility and labeling reactions?
Should one remove 1 M imidazole from the sample after elution from a nickel column? In many
cases it is good to have (an) additional purification step(s) following the elution of the sample from
a Ni column. Size exclusion chromatography (SEC) is a convenient and effective way to remove
the imidazole. If one finds that imidazole is an important variable in maintaining sample
homogeneity, one can also simply reduce the concentration of imidazole from 1 M to a more
reasonable level (0.02 – 0.2 M) for inclusion of crystallization reagents. In some cases a protein
might not crystallize in the presence of imidazole. To remove the imidazole one can try
precipitating the protein in ammonium sulfate and resuspending the sample in low salt buffer and
purify by ion exchange or SEC. One can also remove imidazole by dialysis. In one instance
(personal communication from Artem G. Evdokimov) decent crystals could only be obtained using
0.6 M imidazole/acetate buffer and two imidazole molecules were seen occupying hydrophobic
spots on the protein surface. Sometimes the reason Ni column purified proteins precipitate without
imidazole is that the Ni ions leak from the Ni column. The presence of imidazole may have been
chelating this excess and trace Ni and once removed the remaining Ni Ni may cause the protein
with a His tag to aggregate in solution. One may be able to remove this excess Ni using a Ni
chelating resin (Hampton Research catalog number HR2-312). Or one can leave the imidazole
buffer with the sample in a more reasonable concentration (0.02 – 0.1 M) or try citrate buffer
(chelator) or EDTA. If you are working with a metalloprotein and need metals in the sample, using
the chelating resin rather than leaving a chelating reagent in the sample is obviously a better choice.
What to do when protein is getting precipitated during dialysis?
Evaluate how different protein concentrations influences the precipitation of the sample after
storage. Generally it is better to store proteins more concentrated than dilute. Losses due to
adsorption will be relatively less when the sample is at 10 mg/ml than at 0.5 mg/ml. However, if
storing the protein too concentrated leads to precipitation, store the protein at a dilute concentration
and then concentrate the sample immediately after thawing and right before crystallization
experiments.
In some instances a protein will crystallize during a freeze thaw so be sure to check the precipitate
under magnification for the presence of microcrystals.
Try warming a precipitate protein by holding or rubbing the tube in your warm hands or holding
the tube under a stream of warm (approximately 37 degrees Celsius) tap water with gentle swirling.
Be gentle. Do not shake or vortex. Avoid foaming
Which reducing agent do you prefer?
TCEP HCl, DTT and BME have been shown to reduce protein aggregation that may inhibit
crystallization.Reducing agents can interact with metals within your sample resulting in poor
efficiency of your reducing agent. If possible it is suggested to use a metal chelating agent such as
EDTA to remove all undesired metals. Unfortunately, this may not be possible since many
enzymes require metal in order to properly function. BME is especially sensitive to copper, cobalt
and many phosphate buffers. DTT is sensitive to nickel therefore caution should be used when if
used in combination with affinity chromatography.
DTT has a shorter half-life than BME but has a low volatility. Both have a big dependence of pH.
At pH 6.5 (20ºC) BME has a half-life greater than 100 hours and DTT 40 hours. At pH 8.5
(20ºC) half-lives reduce to 4 h and 1.4 h respectively (Stevens et al., 1983).

Why is my protein acting differently at the same pH?

HEPES Stable pH vs. temperature, no primary amine groups, no metal chelation, near physiologic
pH range. HEPES is often used to maintain protein solubility in biochemical experiments.The
activity increased in the following order: citrate < Tris = potassium phosphate< HEPES. presence
of more than 10 mM HEPES at pH 8.0 was attributed to the surfactant activity of this buffer,,
HEPES and Tris, being positively charged at pH 7.0, were not bound as readily as phosphate.
oligonucleotide initiator decreased in the following
order: cacodylate> MES >HEPES> TRIS >phosphat.The presence of as much as 100 mM NaCl
increased the stability and reversibility of unfolding transitions in Hepes buffer but not in
phosphate buffer at pH 7.4
HEPES interferes with the Lowry protein assay (not the Bradford assay). It can form radicals under
various conditions and should not be used in systems where radicals are being studied.
Tris possesses a potentially reactive amine and participates in various enzymatic reactions. The
pH of a Tris buffer is affected by the temperature (see above) and the concentration. The pH
decreases 0.1 unit upon a tenfold dilution.
What is the relationship between the protein beta sheets and structure stability?
Beta sheets are stabilized by hydrophobic contacts and backbone hydrogen bonding. Alpha helices
are largely stabilized by backbone hydrogen bonding. That is, local interactions dominate in a helix,
whereas a sheet is stabilized by long range contacts. So, a sheet is slightly inferior in terms of
stability.
As mentioned, sheets have more hydrophobic contacts, mutation or misfolding, could result in
exposure of hydrophobic residues. In which case, proteins aggregate to prevent hydrophobic
exposure/avoid entropic penalty. folding aggregates, inclusion bodies and amyloid
This is also one of the primary reasons why designing beta sheet peptides is not easy, i.e. they can
very easily aggregate and form amyloids : Page on sciencedirect.com
Both ordered and disordered aggregates show a high/higher beta sheet content. So, the energy
barrier for helix---> sheet transition is higher.
The solubility of disulfide bond containing protein can be increased by using a host strain with a
more oxidizing cytoplasmic environment. Two strains are commercially available (Novagen):
AD494, which has a mutation in thioredoxin reductase (trxB).
Origami, a double mutant in thioredoxin reductase (trxB) and glutathione reductase (gor).
Why is my protein acting differently at the same pH?
HEPES Stable pH vs. temperature, no primary amine groups, no metal chelation, near physiologic
pH range. HEPES is often used to maintain protein solubility in biochemical experiments.The
activity increased in the following order: citrate < Tris = potassium phosphate< HEPES. presence
of more than 10 mM HEPES at pH 8.0 was attributed to the surfactant activity of this buffer,
HEPES and Tris, being positively charged at pH 7.0, were not bound as readily as phosphate.
oligonucleotide initiator decreased in the following order: cacodylate> MES >HEPES>
TRIS >phosphat. The presence of as much as 100 mM NaCl increased the stability and reversibility
of unfolding transitions in Hepes buffer but not in phosphate buffer at pH 7.4
HEPES interferes with the Lowry protein assay (not the Bradford assay). It can form radicals under
various conditions and should not be used in systems where radicals are being studied.
Tris possesses a potentially reactive amine and participates in various enzymatic reactions. The
pH of a Tris buffer is affected by the temperature (see above) and the concentration. The pH
decreases 0.1 unit upon a tenfold dilution.
What to do when protein is getting precipitated during dialysis?
Evaluate how different protein concentrations influences the precipitation of the sample after
storage. Generally it is better to store proteins more concentrated than dilute. Losses due to
adsorption will be relatively less when the sample is at 10 mg/ml than at 0.5 mg/ml. However, if
storing the protein too concentrated leads to precipitation, store the protein at a dilute concentration
and then concentrate the sample immediately after thawing and right before crystallization
experiments.
In some instances a protein will crystallize during a freeze thaw so be sure to check the precipitate
under magnification for the presence of microcrystals.
Try warming a precipitate protein by holding or rubbing the tube in your warm hands or holding
the tube under a stream of warm (approximately 37 degrees Celsius) tap water with gentle swirling.
Be gentle. Do not shake or vortex. Avoid foaming
Affects of imidazole on protein solubility and labeling reactions?
Should one remove 1 M imidazole from the sample after elution from a nickel column? In many
cases it is good to have (an) additional purification step(s) following the elution of the sample from
a Ni column. Size exclusion chromatography (SEC) is a convenient and effective way to remove
the imidazole. If one finds that imidazole is an important variable in maintaining sample
homogeneity, one can also simply reduce the concentration of imidazole from 1 M to a more
reasonable level (0.02 – 0.2 M) for inclusion of crystallization reagents. In some cases a protein
might not crystallize in the presence of imidazole. To remove the imidazole one can try
precipitating the protein in ammonium sulfate and resuspending the sample in low salt buffer and
purify by ion exchange or SEC. One can also remove imidazole by dialysis. In one instance
(personal communication from Artem G. Evdokimov) decent crystals could only be obtained using
0.6 M imidazole/acetate buffer and two imidazole molecules were seen occupying hydrophobic
spots on the protein surface. Sometimes the reason Ni column purified proteins precipitate without
imidazole is that the Ni ions leak from the Ni column. The presence of imidazole may have been
chelating this excess and trace Ni and once removed the remaining Ni Ni may cause the protein
with a His tag to aggregate in solution. One may be able to remove this excess Ni using a Ni
chelating resin (Hampton Research catalog number HR2-312). Or one can leave the imidazole
buffer with the sample in a more reasonable concentration (0.02 – 0.1 M) or try citrate buffer
(chelator) or EDTA. If you are working with a metalloprotein and need metals in the sample, using
the chelating resin rather than leaving a chelating reagent in the sample is obviously a better choice.

DTT has a shorter half-life than BME but has a low volatility. Both have a big dependence of pH.
At pH 6.5 (20ºC) BME has a half-life greater than 100 hours and DTT 40 hours. At pH 8.5
(20ºC) half-lives reduce to 4 h and 1.4 h respectively (Stevens et al., 1983).

The solubility of disulfide bond containing protein can be increased by using a host strain with a
more oxidizing cytoplasmic environment. Two strains are commercially available (Novagen):
AD494, which has a mutation in thioredoxin reductase (trxB).
Origami, a double mutant in thioredoxin reductase (trxB) and glutathione reductase (gor).

Since 2011, we have sought to apply a space-crystallization experiment to protein kinase and
repeated our proposals. However, at the ground test under the condition of 20℃, stable
 crystallization failed to reach that of a space experiment. Under 20℃, kinase proteins agglomerate
and precipitate rapidly; therefore, we conduct experiments under 4℃ to suppress agglomeration.
JAXA now offers the opportunity for conducting a crystallization experiment at 4℃, thereby
opening the door to space experiments involving unstable proteins. Although we have yet to
conduct X-ray diffraction analysis, judging from the appearance, we consider the crystal to be of
high quality and are satisfied with the experiment.
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