C-terminal Cleavage During Overexpression - (Jan/25/2011 )

Hi,

I'm currently using E. coli to express a fusion protein, but my fusion protein keeps getting cleaved on the C-terminus at various points. I know this because I can overexpress and purify the isotopically-labeled fusion proteins, separate the fusion proteins of various lengths with HPLC, and then use NMR to see that the C-terminal residues are missing. The solubility tag I'm using works so well that I can't get the fusion protein to express into the inclusion body to try and prevent cleavage.


Does anybody know if there is something that I can add to my cultures to prevent C-terminal cleavage? I'm trying to avoid making a new fusion protein construct.


Thanks!!!!

connoc4 on Tue Jan 25 21:40:12 2011 said:

Hi,
I notice similar problem with my protein. I am over-expressing this protein in E.coli with a N-terminal SUMO-tag (12 kDa). The cleavage is happening near N-terminal end of protein of interest (not inside the SUMO-tag region). As seen on the gel, about equal amounts of full-length and truncated version (N-terminal cleaved) are produced at the time of over-expression. I never tried expressing the protein in to inclusion body. Always I wanted to express it into soluble fraction. Let me know if you find any solution.

thanks
-rbrgouda

you may try a carboxypeptidase inhibitor in your medium when you induce your protein.