by S Patnaik, September 2004
The stability of peptide bonds ensures that the primary structure of proteins remain intact under biological conditions. Of course, proteases in the environment can cleave the bonds and break a protein apart. Long term chemical changes to proteins include deamidation of asparagine (alkaline pH and phosphates in buffers promote this), oxidation of thiol- or aromatic ring-containing amino acids (usually by heavy metal ions), beta elimination, etc.
However, the tertiary structures of proteins (which largely determine their functions) are vulnerable to changes in their environment. The chances of adversely affecting proteins increase as they are manipulated more (purification, freezing, thawing, ...). Proteins at 4 deg usually have shelf-lives of months.
The first hint that a protein has stability problems is the appearance of preicipitates in the protein solution. Such precipitation can occur over long term even if precautions have been taken to avoid acute stresses (like freezing and thawing).
1. Always wear gloves and use clean tubes and pipet tips - protect protein solutions from contamination with proteases and chemicals in the environment.
2. Proteins need to be stored at a high level of concentration - atleast 1 mg/ml. This may not be realistic in some cases and so, if possible, an inert protein such as BSA should be added to raise the total protein concentration to 10-15 mg/ml.
3. Protein solutions should not be vigorously shaken by vortexing. That not only generates bubbles and raises oxygen levels in the solution, but can also affect protein structure because of the high shearing forces.
4. The range of temperatures that a protein goes through during freezing and thawing exposes it to extremes of pH and salt. To lower this degrading effect during freezing, protein solutions should be snap frozen by immersing tubes in dry ice and ethanol/acetone mixture (even for -20 deg. storage). Rapid thawing can be performed by immersing tubes in a beaker of water at room temperature or by holding a tube in a stream of water. One should still aliquot solutions to avoid multiple freezing and thawing.
5. One needs to be careful about the buffer used to store a protein in. For enzymes, storage buffers ideally are the same ones in which the enzyme shows good activity. Similarly for pH although it may need to be different if the optimal pH is unsuitable for downstream applications like chromatography. Phosphates in some buffers can inhibit certain enzymes. Some proteins require certain ions like magnesium and/or certain concentration of NaCl for optimal stability. At the same time even 150 mM NaCl can promote aggregation of certain proteins. Reducing environment, because of low pH and reducing agents is usually detrimental to proteins that have disulfide bonds (usually lumenal and extracellular proteins). However, they are usually needed (1-5 mM) for proteins that have free thiol groups, usually intracellular proteins. Some proteins can also be more stable when their ligands are present in the solution. E.g., galectin proteins retain functionality when stored with lactose.
6. Heavy metal ions can be damaging to many proteins, primarily by oxidizing thiol groups. Reducing agents like DTT and/or 0.1mM EDTA can help in preventing such damage.
7. Protease inhibitors reduce damage from proteases but they can be toxic to cells. In some cases they can also inhibit certain protein activities.
8. Stabilizers like glycerol or ethylene glycol - 20-50% - and sucrose - 0.3 to 0.5 M - help proteins retain their activity during freezing and thawing. Polymers like PEG and polysorbates can do the same but at much lower concentration. Such solutions never solidify upon freezing (-20 deg) and thus can be used many times without a need for thawing. However, the high viscosity can negate their use if certain downstream procedures, like chromatography, have to be done.
9. Lyophilization is a useful process for long term storage. However, the protein sample needs to be rapidly frozen before lyophilizing. In some cases, lyophilized proteins may not readily resolubilize (suggesting denaturation) - thus pilot tests should be performed.
10. Some studies have indicated that 50mM each of arginine and glutamic acid in solution significantly increases protein stability (and solubility) without adversely affecting protein activity.
11. Azide (sodium) in the storage solution - 0.02-0.05 % - or thiomerosal - 0.01% - can prevent microbial contamination of the solution - especially important for 4 deg storage.
12. Protein solutions can also be stored as 'salted-out' with ammonium sulfate (usually 70% saturated). even at 4 deg., such salt-outs are stable for many months.. The salt can be removed by dialysis.
13. Citrate, tris and histidine buffers are less likely to undergo pH changes during freezing/thawing. Sod. phosphate buffers however undergo big changes.