We have expressed and purified the recombinant viral protein found in the insoluble fraction. Please suggest some methods or reagents to solubilize the protein.
The hard task is solubilizing it without denaturing it. The addition of mild surfactants can help with some proteins. Sonication may help.
If the protein is in the insoluble fraction because it did not fold properly, which often happens, then it will probably be necessary to completely denature it with concentrated urea or guanidine-HCl, then refold it using any of several techniques. As this process can be difficult to achieve without several experiments, it is often worth spending some time to try to avoid the problem in the first place.
Sometimes, a portion of the expressed protein can remain in the soluble fraction if the expression conditions and/or the expression strain are changed. One approach that sometimes works is to lower the expression temperature. as low as 16oC. This slows down expression, possibly allowing time for the some of the protein to fold properly instead of aggregating.
In some cases resuspending the purified inclusion bodies in buffer with 2 M Urea, freezing at -20 C over night and thawing can bring the proteins into solution. Sometimes.....
Try out refolding of insoluble fraction using 2 M urea/ 6 M Urea and then proceed for refolding by using appropriate refolding buffers at PI of protein of interest.
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