Protein Storage For Protein Stability And Less Protein Degradation

Protein Storage and Stability

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. Although the primary structure of the protein comprises of the amino acid sequence, the functional properties of the protein crucially rely on the three-dimensional or tertiary structure. Similarly, protein modifications for e.g., glycosylation, phosphorylation, may change the properties and function of the protein. 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. Some proteins are relatively rigid hence can function as structural meshes or connective lines. Proteins with reversible conformational changes (polymerization or depolymerization) are crucial for protein-protein interaction, growth and the transmission of information from cell to cell or within the cell.

Why is the stability of proteins essential?

With superfluous structural and functional variations, proteins require specific conditions for their conformation, stability and activity. Unfavourable conditions or sudden change in the native environment leads to protein degradation, denaturation or precipitation, which are mostly irreversible changes. Isolation of purified proteins is a tedious task and needs to get customized according to the protein of interest. However, it is essential to understand the behaviour of the specific protein prior to the development of an assay for its purification. 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. These factors may cause denaturation of the protein, subsequently alter activity profile or lose the biological function or aggregation of the protein. Hence, to maintain the functional properties of the protein intact, it is necessary to mimic in-vivo conditions as closely as possible in the in-vitro purification or storage. The factors that affect the stability of proteins on a long-term basis are:

1. Clearing: As the protein gets released into the buffer, cellular debris and other precipitated material should be removed by either filtration or centrifugation.
2. Buffers: The protein stability also relies on the storage pH provided by the buffer. Hence it is essential to provide proper pH environment to ensure optimum stability. If the protein is used for the biological assay for e.g., enzyme activity or in-gel activity assay, the buffer and pH should be set in a way that it does not hamper the functional conformation of the protein. For long term storage as well, it is essential to choose a correct buffer which in ideal condition should never interact with the protein. The buffer may require additional salt for protein activity, such as Magnesium, calcium, or metal ions. The ionic strength of these salts must be optimised before storage or purification of the protein. Pierce provide a Protein Stabilizing Cocktail (Product No. 89806) as a 4X solution that promotes storage of proteins at 4°C or -20°C for a longer duration compared to Tris-buffered saline or Phosphate-buffered saline.
3. Reducing environment: Most of the proteins contain a substantial amount of free thiol group containing amino acid, cysteine. Oxidation of these free thiols leads to the formation of a disulfide bond between cysteines. Certain proteins require free thiol groups in their active site, hence, oxidation can lead to the biological activity lose. Majority of the intracellular proteins require free thiols for their function. 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.
4. Metal ion contamination: Heavy metal ions have adverse effects on the activity of proteins. Hence, a chelating agent (0.1 mM-0.2 mM concentration) must be added in the storage buffer to eradicate metal ion contamination, such as ethylenediaminetetraacetic acid (EDTA). Although if the protein activity requires the presence of divalent metal ions, these chelating agents should be avoided.
5. 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. The membrane-associated proteins are usually less soluble in hence they require additional hydrophobic additives such as ethylene glycol or glycerol for proper solubilisation and activity. For long-term storage, glycerol concentration can be kept till 50%.
6. Temperature: Typically, proteins are stable at low temperature, around 4°C. Similar to the pH, proteins have different temperature optima based on the experimental requirements. Low temperature has adverse effect on the active harvest of the protein in purification methods such as chromatography.
7. Proteases: Proteolytic enzymes or proteases break the polypeptide/proteins into smaller fragments hence cause protein precipitation or loss of function. During the preparation of protein sample, the proteolytic enzymes are released into the buffer along with the pool of proteins. Hence, it is required to reduce or inhibit their activity to save the proteins from degradation. Aside from low temperature and rapid purification steps, inhibitors can also be added to the buffer to ensure stability of proteins. These protease inhibitors interact with the active site of the proteases and hamper their proteolytic activity. Generally a protease inhibitor cocktail is added in the buffer solution to inhibit proteases.
8. Additives: To lengthen the stability and shelf life of the protein solutions, certain compounds may be added, which depends on the experimental use and the properties of the proteins. In case of purified antibody protein for Western blotting or immunoprecipitation, cryoprotectants such as glycerol or ethylene glycol (w/v 25% to 50%) needs to be added to refrain the solution from freezing at -20°C. Commercially available multicomponent cryoprotectants can be used to enhance the life of antibodies in storage. Pierce offers SuperFreeze™ (Product No. 31503) and Guardian™ (Product No. 37548) Products as antifreeze agents. SuperFreeze™ Peroxidase Conjugate Stabilizer allows long-term storage of horseradish peroxidase (HRP) conjugates at -20°C while Guardian™ Peroxidase Conjugate Stabilizer/Diluent allows long term storage of peroxidase conjugates in diluted form (as low as 10 ng/ml) at room temperature or 4°C. A lower concentration of Tween-80 (0.01%) reduces the probability of the binding of proteins to the storage tube. Antimicrobial agents such as sodium azide (0.02% to 0.05% w/v) or thimerosal (0.01 % w/v) may be added to prevent microbial growth. Although these additives may enhance the shelf life of the proteins it should be carefully considered depending upon the experimental need as high viscosity may compromise with the quality of the sample as well as the downstream steps.

Where to store protein samples?

Proteins, like any other biological reagent, are sensitive to rough handling. 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. Different storage methods can be followed depending on the experimental demands.

Figure: Rate of protein loss during storage at 4°C. Proteins: (mEGF) murine epidermal growth factor (EGF); (rhEGF) recombinant human EGF; (a.lact) α-lactalbumin. (Source: Richard J. Simpson, Stabilization of Proteins for Storage, 2012)

Quick tips:

1. Aliquot the protein into small tubes to avoid repeated freeze thaw and contamination.
2. Dilute proteins tend the bind with the storing tubes. It is advised to either concentrate them to >1 mg/ml or add a filler such as BSA.
3. Caution should be exercised while handling protease inhibitors as they are toxic.
4. Always follow the data sheet of the protein (especially antibodies) before reconstitution or storage.

References:

1. Deng J, Davies DR, Wisedchaisri G, Wu M, Hol WG, Mehlin C. An improved protocol for rapid freezing of protein samples for long-term storage. Acta Crystallogr D Biol Crystallogr. 2004; 60(1):203-4.
2. Matejtschuk P. Lyophilization of proteins. Methods Mol Biol. 2007; 368:59-72.
3. Storage of purified proteins. Protein Expression and Purification Core Facility, European Molecular Biology Laboratory.
4. Protein stability and storage. Pierce Biotechnology, Inc., 2005.

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