We have been using the Baculovirus system for protein expression. When we purify protein from Hi5s, we have to change the pH from about 6.3 or less to 8.0 for purification. When we change the pH some of the protein precipitates out of solution so we cannot purify it. does anyone have any advice to help stop this? We have tried changing the pH of the Hi5 media, which just makes the cells unhappy and take forever to grow. Anything would be helpful!
Thanks :)
How important is it that the pH is 8.0 for the purification? This may be the pI of the protein. Can you use 7.5 or 8.5?
The salt concentration may have an effect on the solubility of the protein. Try increasing or decreasing it.
Hi there,
As Adam mentioned, the pH change may go across the pI of the protein. Have you calculated the theoritical pI of your POI? Try to avoid the pH being too close to the pI value because it is a destabilizing factor.
What is the pI of your protein? Making sure you are not too close to the pI, as mentioned by Adam above, should help. Generally, the farther you move from the pI, the more soluble your protein will be (within obvious constraints). Also, different buffers in general could help. You can try different salts and concentrations of the salts (higher and lower). Any additives that would help stabilize the protein? Lastly, is it a really insoluble protein to begin with? You may want to consider a solubility tag.
Also- what kind of purification are you planning on putting it through?
You describe that some of the target protein precipitates but apparently not all of it. I assume you're using Hi5's for the expression level advantage and then Express 5 serum free media? Is the expression level relatively high with no protease? At pH 8 the target may not be insoluble but just less soluble which is dependent upon the concentration and forms of the recombinant product.under these conditions. What if there are various glycosylated forms, non-covalent aggregates, internal clips all with various solubilities? If you want to capture all product related forms and keep it all soluble at your pH then a slight amount of dimethyl urea or dimethyl sulfone (not sulfoxide) could keep the material in solution without denaturation or affect on downstream unit operations. Did the gel of the precipitate show additional proteins, perhaps contaminants the precipitated with occlusion of product from media?
Hi everyone,
Thanks so much for all of the answers so far!
The pI of the protein is 6.5 (theoretically). We have been using a Ni-NTA Agarose column from Quiagen for purification of the protein which is 6x His-Tagged. After purification, most of the protein has precipitated out, which I have seen through western blot of the precipitate. I have enough protein after purification for western which is super sensitive, but I cannot pick up any protein with a general protein stain, even with protein stains which are supposed to be more sensitive. So, I assume that I dont have enough protein because it precipitates out. Problem is, the precipitated protein will not go back into solution, so I cannot do anything with it. We get microgram quantities through Bradford assay of purified protein, 18 ug is the most we can get recently.
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