I am currrently working on the enterokinase cleavage on the protein trapped by gfp beads. Since the fused protein obtein the sequence of gfp and flag, this protein is enzyme, when eluting the protein from beads by acid which is of high efficiency, we found the protein enzyme acticity is influenced so that I plan to use a milder way which is enzyme cutting. However, after enterokinase (from NEB) treatment, the target protein length is less than the desired. It seems that enterokinase may have other cleavage site duo to protein substrate conformation. I want to ask how to optimize this process or if you have any experience about this.
Dear Chen Wang
The specificity of enterokinase depends from the presence or not of basic residues esposed in the protein surface that can be cleaved. Theoretically if you are able to identify which is the aspecific cleavage site, you can try to perform mutagenesis of your protein but it is a long work and may alter the properties of your protein.
I tihnk that the easiest solution is replace the enterokinase digestion site, with the TEV digestion site (which is much more specific) and try again.
Since both digestion sites are short, you can easly replace it performing a cicle of PIPE cloning similarly to what was done in the following link:
https://proteocool.blogspot.com/2021/09/tips-of-week12-rapid.html
for N-terminal signal peptide sequence.
best regards
Manuele