Hi,
I am trying to purify a truncated part of protein that needs a proper post-translational modification, thus I use Flag-tagged mammalian construct and try purification with Flag agarose beads. However after overnight incubation with TEV, my protein stays insoluble (still appear in resin-boiled samples), even though it seems like there was a shift of band that suggest TEV cleavage worked.
My questions:
1. If I want to optimise salt concentration in buffer to increase solubility, what should I consider to go higher or lower salt conc?I am now using buffer with 500mM NaCl.
2. Has anybody used Flag-MBP-tag for mammalian protein purification and is it worth try to insert MBP to my construct to increase solubility? I read somewhere that MBP tag can increase solubility, but usually for E.Coli system (though there are modified MBP for mammalian). Is it feasible to use double-tag or better use only MBP-tag for this?
3. I am also try to elute using 3xFlag peptide as alternative. Is there any other easier solution to elute my protein from the beads?
NB:
I don't need super high conc purified protein as I am using them only for protein-protein interaction analysis, not crystalisation.
Any suggestions will be very helpful. Thank you very much!
If the part of the protein you expressed is from the interior, it might be very hydrophobic. It might assume a structure to hide as much of the hydrophobic part as possible. This structure then may not be optimal anymore for your purification procedure.
In the distant past, I had to work with viral core proteins. Some of them were so hydrophobic, it was almost impossible to keep them from precipitating.
Achim Recktenwald
Thank you very much for your insight.
Yes you are right, this part of the protein I am trying to purify is quite structured with a lot of folding needed and thus I didn't consider use bacterial system to express and purify because I knew they will need a proper folding and post-translational modification. I think it could also be the reason why nobody else working with this exact domain hasn't been successful in determining its crystal structure; precipitation seems unavoidable.
In that case, I will consider different approach to analyse its interaction, probably trying to purify them (and try direct protein-protein interaction) is not the most feasible way for now.
Thank you!
Sebastian Schmitt
Thank you very much for your reply!
1. I will try to optimise the buffer condition based on your suggestion. To be honest I just tried with the usual buffer people use before (not for this particular part of protein) and didn't consider checking the pI before. So, thank you for this suggestion.
2. Yes I also read that MBP tag is quite big, and that was also one of my concern in using them. If it will likely interfere with the proper folding of my protein (which needs a lot of folding), that could be a big issue...
3. Yes I tried TEV but that was when I have the issue with precipitation. I thought still keeping the Flag-tag (at least I can still do protein-protein interaction analysis with the tag intact) will solve the problem of precipitation, thus I am considering Flag peptide.
In summary, you should try a more physiological buffer; you might have to change the construct (FLAG keeps it soluble, thus, keep some more AA on your construct) and the tag (strep tag purification is cheaper) and maybe you could do the interaction study with a tag on the construct. --> These are excellent suggestion, I will try it.
Thank you very much!
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