When purifying baculovirus expressed GST-tagged proteins from Sf9 cells by affinity chromatography I do quite frequently co-purify endogenous insect GSTs with my recombinant protein. Even while these contaminations can be removed during the purification process by various more or less laborious methods, I wonder if anybody has an idea how to generally reduce these endogenous GSTs by either modifying the culture conditions of the Sf9 cells or by some kind of fast and efficient pre-clearing step.
The only way I know of is to purify it away. Cells need adequate levels of GST. We usually use SP or Q sepharose as a first step in our purifications depending on the charge of the fusion protein. Perhaps your fused protein will has a different pI than GST alone.
Thanks, Michael for your comment. I have tried such approaches successfully for some proteins, but as you wrote correctly, they have to be optimised for each new fusion protein separately. I still hope to find a way to clear either lysate or cells just before purification of the endogenous GSTs by some general method to obtain purer preps without the need to polish individually.
Best way would be to use two tags and then performs two purifications. as you already have expressed your protein, you can try to use different size filters before purification, if you the contaminating proteins are or different sizes than your protein of interest.
Another naive thought I have is, if your peptide is stable enough on its own, you can cleave the GST tag after the first purification and pass the GST affinity media again (or do a on-column cleavage to avoid doing it twice).
I know this is probably not a good way if you don't want to cleave it anyway, but just another thought of mine.
@ Manoj and Huanyao, thank you for your suggestions .Both approaches work fine with a lot of proteins in my hands. Especially the cleavage of the tag by 3C protease frequently works great if one uses GST-tagged 3C with a GST-tagged recombinant protein, so cleavage and protease removal can be done in one step. And tag-free proteins are in many cases desirable anyway. It would nevertheless be nice to find conditions under which insect GSTs are not bound to the affinity matrix in the first place, since every additional purification step reduces protein yield and in many cases also activity of the respective protein.
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