I am currently refolding the NS1 Dengue protein in a refolding buffer of PBS 1X pH 7.2 + 30% sucrose (protein stabilizer). I dissolved the inclusions with 1X PBS buffer pH 7.2 + 8M Urea + 30% sucrose.
However, protein always precipitates after just a few hours, causing the concentration to only 0.1mg/ml
The pI of NS1 protein is 6.2
Can you give me advice on how to reduce protein aggregation?
Any way I can try, except using Arginine which I have tried according to some articles but in fact NS1 still precipitates
Typically, refolding proteins from 8 M urea by dialysis is done by gradually reducing the urea concentration. For example, dialyze the protein from 8 M into 6 M, then 4 M, then 2 M, then 0 M. Don't go in one step from 8 to 0 M. Including arginine (commonly) at 0.5 or 1 M can be helpful. This method uses up a lot of chemicals and takes a long time. The protein solution shold be as dilute as practicable to reduce aggregation. The dialysis bag or cassette should be stirred as fast as possible.
Another common method is to very slowly drip the protein solution in 8 M urea into a rapidly stirred refolding buffer without urea, such that the final concentration of protein is less than 30 µg/mL. The idea is to dilute the protein very rapidly so molecules don't have a chance to aggregate Then, you are faced with having to concentrate a large volume of dilute protein. Again, including a high concentration of arginine in the refolding buffer is useful.
A third method of refolding is to bind the protein in 8 M urea to a column (e.g. an IMAC column using a His tag), then run a slowly decreasing urea gradient prior to eluting the protein with imidazole. Again, include arginine in the buffers.
Whatever method you use, you are likely to end up with a mixture of monomeric protein (hopefully) and aggregated protein. To remove the aggregated protein, you can run the protein over a gel filtration column.
- Badges
- Science method
- Similar topics
- Biological Science
- Ecology