I express antibody fragments using SUMO tag, in E. coli. I have been able to get purified proteins after couple steps of chromatography, but there is a very strong band of lower size after IMAC. We have analyzed that particular band by mass spectrometry and it seems to be our protein degraded (histag + SUMO tag + beginning of the protein; the presence of histag would explain why it's there after IMAC). I use mild sonication conditions (2seg ON/4seg OFF, 30% amplitude, 10min total sonication on ice) to avoid degradation and also protein inhibitors.
Does anyone have an explanation/hypothesis to explain why this SUMO-tagged protein is being cleaved?
Based on MS, the cleavage is not on the enzyme's cleavage site (ULP-1 protease, to remove the tag).
Thank you Iqra Bano
for the comments. Yes, I take care to always keep everything chill and I do the whole process on ince. The pH seems to be fine and I think I'm not even sonicating enough, since I still see some bacterium pellet after centrifugation, but the cleavage is still there. It's weird because it seems to be on the same amino acid position, since the band is always at the same size.
Sometimes a protein has a protease-hypersensitive site. One way to reduce the amount of proteolysis in such cases is to use an expression strain that is lacking certain proteases. Another way is to introduce a site-directed mutation at the hypersensitive site.
Interesting, Adam B Shapiro . Do you use any tool to predict these sites based on the protein sequence?
Here is a predictor based on amino acid sequence.
https://web.expasy.org/peptide_cutter/
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