Dear Hadi

Generally my firts choice for tag cleavage is TEV protease, not only because is cheap (you can produce it by your self) but because if work very well in standard buffer. Sometimes i used SUMO and Fatt.Xa protease and they worked well to me in standard conditions (respect to your buffer i just included 2mM of DTT in the buffer).

However my experience with TEV, suggest me that some times the partial cleavage of a protein with the protease could be associated, not to the protease activity, but to the low exposition of the digestion site in the fusion due to the protein unfolding or protein aggregation. Did you check by SEC the aggregation state of your purified fusion before digestion?

High molecular weight aggregates are difficult to be cleaved. In this case you can try te reduce aggegation or degrasing the concentrarion of your proteins or adding in your reaction some detergents, also if  how you can see in the attached file SUMO seems not is the best to use in presence of detergents.

good luck

manuele

Hi Hadi,

SUMO protease is a robust and highly active enzyme that works in nearly any buffer condition; the enzyme shouldn't be an issue. The lack of activity could be due to the construct design. SUMO protease activity is hindered if the first amino acid in the native protein is proline, aspartic acid, glutamate, valine, isoleucine or leucine. Check your construct first.

@

Nicholas Spellmon

Hi Nicholas

I checked out the sequence of protein and I know that sumo protease can cleave Gly-Gly motif. I sure that some amico acid which you mentioned are not exist in sequence of protein.