I'm working with recombinant protein, it's a protease enzyme that we use E. coli to synthesize, and usually, it's stable for up to six months in the -80 freezer. Recently, the protein activity has been acting weird or showing a sudden drop after just two months, knowing that this enzyme is not expected to digest itself. Does anyone have an explanation, or even know how to solve this problem?
Thanks
Hi,
I think that it is possible with the following reasons:
1, Protein degradation
The enzyme may not digest itself but the presence of proteases or/and contaminants in the E.coli lysate could degrade the protein over the time. As a consequence, the activity of the enzyme could decline.
In case of the presence of proteases, you could consider adding protease inhibitors during protein purification to prevent proteolytic degradation.
2, Thawing
The loss of protein activity as well as denaturation can occur if the protein is frequently freeze-thawed. I normally aliquot the protein into many tubes with smaller volume before freezing.
3, Variability between batches
It is possible that protein expression after purification may be variable between batches, as a result, protein activity will be different over the time.
You can minimize the variability by maintaining consistency in protein purification procedures.
4, Aggregation of protein
Protein activity can be influenced by its aggregation occurring over the time because of several factors such as pH levels, salt-buffed solutions, and high protein concentrations. To minimize this, you can maintain the protein at low concentrations, use appropriate buffers, or introduce chaperones to prevent protein misfolding and aggregation.
I hope this information is useful for you.
Good luck!
One thing I would add is that the way the protein is handled can have a significant impact on its stability. If you are planning to re-freeze an aliquot of the protein for later use, it is recommended that the remainder be flash-frozen, rather than allowed to freeze slowly. This is because when a solution freezes slowly, the solutes have time to separate from the ice. This can result in an excessively high concentration of protein mixed with an excessively high concentration of salts, which could cause a loss of protein activity.
To flash-freeze a protein, it should be in a small volume, such as 100 µl or less, in a microcentrifuge tube. The tube should be submerged in either liquid nitrogen, a dry ice-ethanol bath, or dry ice powder until thoroughly frozen, then placed in the -80.
When the protein is thawed out, it should be remixed by gently vortexing, or by gently flicking the tube with a fingertip. Excessive vortexing can lead to denaturation and loss of activity.
Some proteins do not respond well to repeated freezing and thawing, In such a case, after the protein is prepared, divide it into many small single-use aliquots. Flash-freeze them, but use each one only once, or until the activity starts to decrease.
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