Recently I am expressing one protein in BL21(DE3) and the protein
undergoes N-terminal degradation.
I am trying to keep this crucial N-terminal tail on the protein,
which has MRS at the first 3 positions.
Digging in to the literatures, I found the N-end rule, which tell
that the proteins bearing N-terminal Arg or lys have much shorter half life.
I am not sure whether this is my problem.
It surely is not. An N-end rule has to do with ubiquitination, and it is
absent in E.coli.
However, I want to found one E. coli mutant strain that lacks aat and is
unable to degrade N-end rule substrates that bear N-terminal Arg or Lys.
Don't think such a strain exists and totally not sure this is really your
problem. (Also, what's AAT has to do with it? Is this your requirement for
something else?) Try inhibiting proteases better. E.g., metal-dependent
proteases are a common problem with IMAC-based purification. First thing is
to find whether the degradation occurs inside the cells or during
purification step(s). If the former, why not fuse the thing to an
N-terminal tag, thus only purifying non-degraded N-termini? (Assuming a
monomer, of course). If N-term. has to be native, His-tagged SUMO fusion
cleaved with SUMO protease will leave no non-native residues (assuming none
was introduced during cloning, which is rather trivial to ensure these days).
- Dima
