Hello,
My expressed protein is at 2 M urea (it will precipitate if I take urea out) and I need to cleave its his tag with bovine enterokinase.
I read some references that enterokinase might be active in the presence of between 1-4 M urea, has someone had experience (enterokinase removes his-tag in 2 M urea)?
Additionally, I read that for cleaving the target protein it is advisable to maintain its concentration at 1mg / mL. I could take out urea from the above mentioned sample, but then the target protein concentration could not be above 0.2 mg / mL. In this case, how do you change the contact time with enterokinase?
Agnes
Digest the protein when it is still on the Ni-resin under non-denaturating conditions, collect the released material and dialyze against 2M urea to re-solubilize the protein, concentrate it if needed.
i think the basic good way to find out is to keep the digestion of your protein at different concentrations of urea and find out. Enterokinase(EK) cleaving fusion protein is specific and one need not worry on non specific cleavage. once you establish the protocol for which molar Urea to use, one can try different substrate to Enzyme concentrations. don't bring too many changes in one experiment that you will not be able to trace what the effect of the parameter.
Digest the protein when it is still on the Ni-resin under non-denaturating conditions, collect the released material and dialyze against 2M urea to re-solubilize the protein, concentrate it if needed.
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