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Recombinant Protein Storage Principle and Method

Abstract:     How to effectively store recombinant protein is a problem after purification. The activity of fused protein may be worse or even lost. The purified protein is easily inactivated and denatured without proper storage.
Keywords:   Recombinant Protein Storage, Protein Purification, Protein Activity

1. Protein Storage Principles

Protein storage should follow several basic principles below:

1.1. Low Temperature

It's important to store proteins at low temperature. Low temperature means lower energy level, which can help to slow down thermal motion inside the protein molecule and decrease the possibility of bond cleavage. Besides, microbial reproduction slows down at low temperature. Potential proteinase activity decreases, which help to maintain protein activity.

1.2. Sterilization

It's required to wear gloves during protein assay in order to protect experimenters themselves. In biological assays, proteins are the main protective items. There are some proteinases on our skin which may degrade protein samples. Besides, other chemical substances may also pollute protein samples. It's better to store protein in the sterile container to prevent bacterial growth.

1.3. Concentration

It's suggested to store recombinant proteins in a higher concentration(1mg/ml or above) to improve protein stability. High concentration can help to prevent the adherence of proteins on the plastic or wall of glass tube. It should be noted that hydrophobins are easily adhered to the wall of glass tube.

1.4. Aliquots of Proteins

Proteins require for cryopreservation but are experimentally used at room or higher temperature. Ice crystal produced by freeze-thaw cycle will damage protein structure and conformation. It's suggested to aliquot and store the purified protein to decrease times of freezing-thawing circle. Melt the required protein before use to assure protein sample is subjected to single freezing-thaw.

1.5. Quick Freezing and Melting

Quick freezing method can reduce the exposure time of protein molecule under the conditions of higher salinity or pH. Quick freezing can be achieved by liquid nitrogen. After that, proteins can be stored at -20℃ or -80℃. Quick freezing is also very important and can be conducted in the water.

1.6. Avoidance of Shock

Shear stress produced by shock may damage proteins. During the shock, bubbles generated in the liquid enable the entering of oxygen. Then, proteins are in the oxidizing environment. Since cysteine in the protein is easily oxidized to cystine, this process may damage protein activity.

1.7. Additives

Proteins should be stored in the suitable buffer with some additives to assure the stability. E.g. When stored at -20℃, 10%-50% glycerol can be added to avoid freeze-thaw cycle. Besides, preservatives can be added to prevent bacterial growth. PMSF can be also added to prevent the damage of protease on proteins. Some reductants like DTT can prevent protein oxidation. Selected additives should not affect protein activity.

2. Protein Storage Method

The storage method depends on its stability and when to use.

2.1. Long-term Storage(Six months or above)

Lyophilized recombinant proteins are completely dehydrated and can be stored for several years in the fridge without getting damp. Fused recombinant proteins can be aliquoted and stored for 6 months at -20℃ or -80℃.

  • Aliquoting helps to maximally reduce the damage of freeze-thaw cycle on the activity of antibodies and recombinant proteins. The contamination caused by sucking in the same tube is also reduced.
  • Aaliquot size should follow the required assay volume(10µl for each tube at least). Smaller size greatly affect protein concentration due to evaporation and adherence to tube wall.
  • The rest mother liquid should be stored at 4℃ to avoid refreezing if not all aliquoted recombinant proteins are used.
  • The protein working solution should be prepared and used in the same day. It's better to store at 4℃ within 1 day.

2.2. Short-term Storage(within 1 week)

Most proteins can be shortly stored at 4℃ to avoid the effect of freeze-thaw cycle on activity. However, microbial contamination may happen. If the storage time is above 24h, it's suggested to filter samples with 0.22μm filter membrane and store in the sterile tube. It should be noted that not all proteins can be stored at 4℃.

3. Notes for Using Recombinant Proteins

Upon receipt of recombinant proteins, please centrifuge for 1min at 10,000rpm to bring all lyophilized powders to the bottom of the tube. Then open the tube for dissolving. Fused buffers are usually sterile pure water or buffer required for the next assay step. Gently shake to dissolve proteins and use the tip for pipetting till completely dissolution. Vortex mixer can't be used for rapidly or greatly shaking.

It's suggested to aliquot and store rest proteins at -20℃ or -80℃. BSA can be added for preventing protein degradation as protectant.

REFERENCES

[1]Guidelines to reach high-quality purified recombinant proteins, PMID: 29151158.
[2]A strategy for targeting recombinant proteins to protein storage vacuoles by fusion to Brassica napus napin in napin-depleted seeds, PMID: 24394588.