I am working on a chlamydial protein that appears to work in vivo, but once I purified it is not active. I tried testing it with a maltose binding protein to make it soluble, but I fear that it might hamper the activity. Cleaving the MBP causes the protein to crash however. I have read about adding Urea and guanidine-HCl in my assays, but I am skeptical if it will work. Does anyone have experience with this issue?
Sorry, but your description is very cryptic. Can you provide some specifics? Is it an enzyme?, membrane protein? Is the MBP fusion protein active? How does your protein "crash"?
Yes I can. Sorry, I am new to research gate and I was not sure how to edit. So, it is an enzyme.
The fusion is soluble, but it appears to not be active. I use a factor xa cleavage because I was not sure if the fusion tag was interfering with enzymatic activity.
It is suspected to work due to data from a gene deletion and rescue experiment (not sure if this is the correct term). The experiment, in which you add a plasmid with protein of interest into an organism that has a related gene required for cell survival deleted.
So, I use the term crash because, without the MBP (after a prolong Factor Xa cleavage), a white precipitant is formed. If I pellet the solution, and resuspend the pellet in solution and run a get, it is my protein of interest.
I have been fighting for a while now with this protein. Even with the MBP fusion, a high percentage of the protein will separate with the insoluble fraction of cell lysis during purification. I utilize high salt, long induction, low temperature conditions.
Using just cell free extract, it appears to be more active than cfe without the protein, but once I try purifying it, I see no activity.
To make it even more of a hassle, I see bands bands of both my MBP fusion, the MBP tag and the protein, so something is cleaving it in the cell prior to purification (I utilize protease inhibitors). So, this reason alone, makes me curious if the only active when cleaved.
Interesting. So your MBP fusion construct rescues the bugs but the purified MBP fusion protein has no activity?
Does this enzyme have an active site inhibitor or active site label that you could use to tag the MBP-enzyme fusion before doing the purification?
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