How to stabilize proteins?
Adding 25-50% glycerol or ethylene glycol can help to stabilize proteins by preventing the formation of ice crystals at -20 °C, that can destroy protein structure, enabling repeated use from a single stock. Protease inhibitors interact with the active site of proteases that hamper proteolytic activity detrimental to protein stock stability.
How to optimize the properties of proteins for pharmaceutical applications?
The pharmaceutical and PHARMACOKINETIC properties of proteins can be optimized by different approaches — for example, by mutagenesis, chemical modification or by designing specific drug-delivery systems.
How do you prevent protein aggregation?
One popular approach is to stabilize the protein and thereby reduce access to partially folded conformations favouring aggregation by hydrophobic contacts, for example. A typical strategy is to add sugars or salts to a protein solution.
What are the stabilizers used to prevent protein aggregation?
Other stabilizers include polyols, PEGs and other polymers that sterically hinder protein–protein interactions and limit diffusion. Free amino acids are also often used; arginine is particularly good at preventing aggregation during the refolding of proteins from inclusion bodies 102.
What affects protein stability?
Many factors affect the process of protein folding, including conformational and compositional stability, cellular environment including temperature and pH, primary and secondary structure, solvation, hydrogen bonding, salt bridges, hydrophobic effects, van der Waals (vdW) forces, ligand binding, cofactor binding, ion ...
What causes protein stability?
To maintain a protein's proper folding and stability, various stabilizing forces act together, including non-covalent interactions (hydrogen bonding, hydrophobic interactions, Van der Waals forces, and salt bridges) under physiological conditions.
Which has the largest influence on stabilizing protein structure?
So, the correct answer is 'Primary-peptide bonds, Secondary-hydrogen bonds, Tertiary-disulfide bridges, Van der Waals interactions and ionic bonds'.
What factors can negatively impact proteins function?
These factors influence the ability of proteins to fold into their correct functional forms. Extreme temperatures affect the stability of proteins and cause them to unfold or denature. Similarly, extreme pH, mechanical forces and chemical denaturants can denature proteins.
What stabilizes the protein conformation?
Folded proteins are stabilized by thousands of noncovalent bonds between amino acids. In addition, chemical forces between a protein and its immediate environment contribute to protein shape and stability.
Which of the following form of protein is most stable?
the most stable protein structure tertiary.the three dimensional structure of the protein is referred to the tertiary structure of he protein.in this structure, the molecules of the protein bend and twist in such a way that it achieves the maximum stability and has the lowest energy state.More items...•
Which of the following is the weakest interaction involved in stabilization of protein molecules?
Hydrophobic interaction is where the H bonds and electrostatic interactions are the weak ones and stabilize the tertiary structure of proteins.
Which of the following can affect protein structure?
The main forces that affect structure are electrostatic forces, hydrogen bonding forces, hydrophobic forces, and disulfide bonds. Each of these affect protein structure in different ways.
How does hydrogen bonding affect protein stability?
(2) The contribution of hydrogen bonds to protein stability is strongly context dependent. (3) Hydrogen bonds by side chains and peptide groups make similar contributions to protein stability. (4) Polar group burial can make a favorable contribution to protein stability even if the polar groups are not hydrogen bonded.
Which of the following could alter or destroy the function of a protein?
Which of the following could alter or destroy the function of a protein? A genetic mutation that changes the amino acid sequence would change the primary structure of the protein.
What causes protein misfolding?
Protein misfolding is a common cellular event that can occur throughout the lifetime of a cell, caused by different events including genetic mutations, translational errors, abnormal protein modifications, thermal or oxidative stress, and incomplete complex formations.
What factors affect the properties of proteins?
Several factors, in addition to the protein per se, affect the foaming properties of proteins--protein concentration, pH, temperature, salt, sugars and lipids.
Why is protein stability important?
Maintaining protein stability not only is necessary in the biochemical purification and spectroscopy, but also is of importance in vivo because of environmental stresses such as water, salts, cold, and heat . Many organisms, fishes, plants, and animals have adapted one common strategy in protecting their cellular proteins against such harsh environmental conditions by accumulating high concentrations of low molecular mass compounds, known as osmolytes. Osmolytes are classified as compatible or counteracting based on their effect on the functional activity of proteins (see, for example, Refs. 11 and 12). Compatible osmolytes (amino acids and their derivatives and polyols) protect proteins against inactivation and denaturation without perturbing the protein functional activity near room temperature. Counteracting osmolytes (methylamines) are built up by organisms to cope with deleterious effects of urea on the functional activity and stability of proteins. Generally, biochemists and biologists store isolated enzymes or organelles in concentrated (~1 M) glycerol ( compatible osmolyte) in order to preserve activity. Timasheff and co-workers 13 investigated the stabilization of protein structure by solvents indicating that osmolytes cause preferential hydration of the proteins. Such stabilizers are sugars, amino acids (glycine, alanine, glutamic and aspartic acids), salting-out salts (Na2 SO 4, NaCl, MgSO 4 ), and glycerol. 13 Perdeuterated glycine was suggested by Leatherborrow and co-workers 14 to facilitate protein NMR spectroscopy of unstable proteins. Byrd and co-workers15 have suggested perdeuterated sorbitol (prepared from glucose) as a stabilization agent for proteins in NMR spectroscopy. It should be mentioned that the addition of glycerol, sugars, and amino acids increases the viscosity of the solution. Thus, the higher thermostability and the possibility of decreasing the NMR line width by using higher temperature and thus decreased rotational correlation times is compensated for by the increased viscosity. The salting-out salts (Na2 SO 4, NaCl, MgSO 4) do not change the viscosity significantly, but lead to problems with sample tuning and heating when high-power decoupling is used. Therefore, use of osmolytes in the buffer is only recommended when it simultaneously suppresses unspecific protein–protein interactions that represent an additional line-broadening mechanism.
Why is protein stability important in 2D crystallization?
Protein stability is a major concern in 2D and 3D crystallization. Many membrane proteins are unstable in detergent solution. The stability often depends critically on the choice of detergent and on the presence of lipids. Strict requirements for a particular detergent or lipid may make 3D crystallization difficult, because either may interfere with crystal contacts. These requirements are easily accommodated in 2D crystallization because the detergent will be removed and lipids are needed anyway for 2D crystal formation. Glycerol is frequently added to enhance protein stability during 2D crystallization, in particular to fragile eukaryotic proteins. Many membrane proteins are much more stable in a lipid bilayer than in detergent solution, as is shown by 2D crystals in which the protein remains stable and active for many months. This is another advantage over 3D crystallization where the protein usually remains exposed to high levels of detergents during crystallization and even in the crystal lattice. 2D crystallization is therefore especially appropriate for sensitive, unstable membrane proteins, in particular for those from higher eukaryotes.
What is the stability of PTEN?
The protein stability of PTEN in the cell is a factor in its signaling properties and PIP3 control. When PTEN C-terminal phosphorylation was discovered, it was also found that there was a correlation between its phosphorylation state and protein stability (Vazquez et al., 2000). This behavior is somewhat paradoxical as the enzymatic activity of phospho-PTEN is impaired which means that the more stable PTEN form is also less functional in PIP3 hydrolysis. Furthermore, the molecular basis of PTEN protein destruction was not yet clear in this early work. Cellular protein clearance often involves Lys ubiquitination which then targets the protein for proteasome-mediated degradation. Ubiquitination is a three-step process involving E1, E2, and E3 ubiquitin transferring enzymes. Several ubiquitin E3 ligases for PTEN have been reported, and the two most extensively studied have been NEDD4-1 (Wang et al., 2007) and WWP2 ( Maddika et al., 2011). NEDD4-1 and WWP2 are both HECT domain E3 ligases and are composed of an N-terminal C2 domain followed by four WW domains and culminating in catalytic HECT domains.
What is the tradeoff between protein accumulation and the health of the plant?
There is always a tradeoff between the efficiency of protein accumulation, the functionally most suitable site of accumulation, and the health of the plant. For example, full-size recombinant antibodies produced in plants accumulate to very low levels if they are targeted to the cytosol.
How do simple proteins form their native conformation?
Many simple proteins fold spontaneously to form their native conformation. In the case of soluble globular proteins, this process is driven by hydrophobic collapse, i.e., the shielding of hydrophobic groups in the core of the protein while hydrophilic residues form the external loops.
How is protein stability modulated?
Protein stability is commonly modulated through variations in solution conditions, a strategy that has relevance in applications to protein overexpression on a small scale, in applications to ligand binding and in applications to commercial formulation in the development of therapeutic proteins .
What is the function of counteracting osmolytes?
Counteracting osmolytes (methylamines) are built up by organisms to cope with deleterious effects of urea on the functional activity and stability of proteins. Generally, biochemists and biologists store isolated enzymes or organelles in concentrated (~1 M) glycerol ( compatible osmolyte) in order to preserve activity.
Why is protein stability important?
Maintaining protein stability not only is necessary in the biochemical purification and spectroscopy, but also is of importance in vivo because of environmental stresses such as water, salts, cold, and heat . Many organisms, fishes, plants, and animals have adapted one common strategy in protecting their cellular proteins against such harsh environmental conditions by accumulating high concentrations of low molecular mass compounds, known as osmolytes. Osmolytes are classified as compatible or counteracting based on their effect on the functional activity of proteins (see, for example, Refs. 11 and 12 ). Compatible osmolytes (amino acids and their derivatives and polyols) protect proteins against inactivation and denaturation without perturbing the protein functional activity near room temperature. Counteracting osmolytes (methylamines) are built up by organisms to cope with deleterious effects of urea on the functional activity and stability of proteins. Generally, biochemists and biologists store isolated enzymes or organelles in concentrated (~1 M) glycerol ( compatible osmolyte) in order to preserve activity. Timasheff and co-workers 13 investigated the stabilization of protein structure by solvents indicating that osmolytes cause preferential hydration of the proteins. Such stabilizers are sugars, amino acids (glycine, alanine, glutamic and aspartic acids), salting-out salts (Na 2 SO 4, NaCl, MgSO 4 ), and glycerol. 13 Perdeuterated glycine was suggested by Leatherborrow and co-workers 14 to facilitate protein NMR spectroscopy of unstable proteins. Byrd and co-workers 15 have suggested perdeuterated sorbitol (prepared from glucose) as a stabilization agent for proteins in NMR spectroscopy. It should be mentioned that the addition of glycerol, sugars, and amino acids increases the viscosity of the solution. Thus, the higher thermostability and the possibility of decreasing the NMR line width by using higher temperature and thus decreased rotational correlation times is compensated for by the increased viscosity. The salting-out salts (Na 2 SO 4, NaCl, MgSO 4) do not change the viscosity significantly, but lead to problems with sample tuning and heating when high-power decoupling is used. Therefore, use of osmolytes in the buffer is only recommended when it simultaneously suppresses unspecific protein–protein interactions that represent an additional line-broadening mechanism.
What is the term for the physical stability of a protein?
The term “ protein stability ” includes many meanings, including proteolytic stability, thermal stability, physical stability (aggregation), et al. Thermal stability is the stability of a molecule at elevated temperatures. Proteolytic stability refers to the resistance toward the action of proteolytic enzymes. Aggregation represents the most vexing physical instability of proteins, which is a process by which misfolded proteins form insoluble precipitates.
How is protein stability modulated?
Protein stability is commonly modulated through variations in solution conditions, a strategy that has relevance in applications to protein overexpression on a small scale, in applications to ligand binding and in applications to commercial formulation in the development of therapeutic proteins .
What is the stability of PTEN?
The protein stability of PTEN in the cell is a factor in its signaling properties and PIP3 control. When PTEN C-terminal phosphorylation was discovered, it was also found that there was a correlation between its phosphorylation state and protein stability (Vazquez et al., 2000 ). This behavior is somewhat paradoxical as the enzymatic activity of phospho-PTEN is impaired which means that the more stable PTEN form is also less functional in PIP3 hydrolysis. Furthermore, the molecular basis of PTEN protein destruction was not yet clear in this early work. Cellular protein clearance often involves Lys ubiquitination which then targets the protein for proteasome-mediated degradation. Ubiquitination is a three-step process involving E1, E2, and E3 ubiquitin transferring enzymes. Several ubiquitin E3 ligases for PTEN have been reported, and the two most extensively studied have been NEDD4-1 ( Wang et al., 2007) and WWP2 ( Maddika et al., 2011 ). NEDD4-1 and WWP2 are both HECT domain E3 ligases and are composed of an N-terminal C2 domain followed by four WW domains and culminating in catalytic HECT domains.
Key Points
Recombinantly expressed proteins are increasingly important in drug therapy. This makes it crucial to assess how their properties as proteins affect drug efficacy, targeting and side effects, as well as the ability to survive long-term storage.
Abstract
The increasing use of recombinantly expressed therapeutic proteins in the pharmaceutical industry has highlighted issues such as their stability during long-term storage and means of efficacious delivery that avoid adverse immunogenic side effects.
Main
During the past two decades, recombinant DNA technology has led to a significant increase in the number of approved biotechnology medicines and a shift away from the production of biologically active materials (biologics) on the basis of animal or human material, and towards cloning and fermentation.
Acknowledgements
D.O. is supported by the Technical Science Research Foundation and the Villum Kann Rasmussen Foundation.
Author information
Department of Pharmaceutics, The Danish University of Pharmaceutical Sciences, Universitetsparken 2, Copenhagen O, DK-2100, Denmark
What factors affect the stability of proteins?
The factors that affect the stability of proteins on a long-term basis are: Clearing: As the protein gets released into the buffer, cellular debris and other precipitated material should be removed by either filtration or centrifugation. Buffers: The protein stability also relies on the storage pH provided by the buffer.
Why do purified proteins need to be stored?
Due to time constraint, the purified or isolated proteins often required being stored for the duration while keeping their activity intact. The extent of storage is remarkably variable and is dependent on the properties of the protein and the storage conditions.
What are proteins made of?
What are Proteins? As one of the multifarious macromolecules, proteins are complex and crucial for cellular functions. Proteins are polymers built of monomer subunits called amino acids connected by a specific type of covalent bond known as a peptide bond. The properties of the protein depend on the type of amino acids present in them. ...
What is the requirement of an additive to get soluble in a buffer solution?
Hydrophobicity: To get soluble in a buffer solution, proteins require variable degrees of hydrophobicity which is attributed by the dipole moment of the amino acids present in the protein. The requirement of an additive varies dramatically with the hydrophobicity of a protein.
How do protein modifications affect the local conformation?
These modifications alter the local conformation and mediate folding or stability as well as drive proteins to different cellular compartments. Proteins also display remarkable variability in terms of structure and flexibility depending upon their folding patterns.
What are the external factors that affect the native conformation of a protein?
The native conformation of the protein can be damaged by many external factors including change in temperature, pH, hydrophobicity, metal ions or mechanical forces owing to the weak interactions holding the three-dimensional conformation of the protein.
What is the reducing agent in a buffer?
While working with intracellular proteins, the buffer must contain a reducing environment to mimic intracellular conditions. Dithiothreitol, reduced glutathione, or 2-mercaptoethanol can be used as a reducing agent in the buffer.
What is the first treatment for a protein that is nascent?
The first treatment of the nascent buffer is either filtration or centrifugation in order to remove cellular debris and other precipitated material.
What temperature is needed for protein stabilization?
As with pH, proteins generally have several temperature optima depending on the experimental context. A good rule of thumb is proteins are more stable at reduced temperature, typically 4°C.
What are proteases inhibitors?
Aside from rapid purification procedures and low temperatures that reduce the protease activity, a class of compounds called protease inhibitors can be added to the buffer. These inhibitors are small molecules that interact with the active site of the protease and inhibit its proteolytic activity. Typical proteases inhibitors include diisopropylfluorophosphate (DIFP), phenylmethylsulfonyl flouride (PMSF), leupeptin, and phosphoramidon.
What are the free thiol groups in proteins?
Proteins containing the amino acid, cysteine, possess a free thiol group, -SH. In the presence of other thiol groups or other cysteine amino acids in the protein chains, oxidation can occur forming a disulfide bond. For proteins that require a free thiol group (e.g., enzymes with a free thiol group at the active site), these oxidation reactions can lead to losses in biological activity. Generally speaking, proteins originally located within the cell will contain free thiol groups. Extracellular proteins generally have thiols in the form of intramolecular disulfide bonds between the cysteines of the protein. These disulfide bonds are usually crucial to tertiary structure and activity. The presence of thiols here can lead to a mixing of disulfides referred to as thiol exchange resulting in denaturation and loss of activity. When dealing with disulfide containing proteins, a reducing environment is detrimental to protein activity, therefore, low pH and free thiols are to be avoided. When working with intracellular proteins containing free thiol groups, a reducing environment that mimics the cell's interior is necessary. Addition of reagents such as dithiothreitol, reduced glutathione, or 2-mercaptoethanol to the buffer is necessary when a reducing environment is required.
