I am currently working on purification protein process. For cleavage of target protein from sumo I applied sumoprotease. the activity of enzyme was too low, could any one recommend me an especial component for increasing the activity? or an especial cofactor for increasing the rate of enzyme reaction?
base buffer for sumo protease is: 50mM tris, 300 mM NaCL, pH 7.5
storage in glycerol 50% (V/v)
Digestion condition is 30C, 100 rpm, over night.
base buffer for cleavage condition is, 20mM tris, 150 mM NaCL, pH 7.8
thanks in advance for your kind attention.
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.
Something that appears to be missing from the buffer composition that you mentioned is the reducing agent and some detergent. The reaction buffer I have used successfully for Sumo protease over the past couple of years is the base buffer that the protein is in (typically 20-50mM buffering agent, 100-500mM NaCl, pH6.8-8.8) supplemented at the time of digestion with 0.2% nonidet (NP40 / IGEPAL CA630 detergent) and 1mM DTT - from memory the DTT is critically required by Sumo protease to be effective. Absolute values for these additives vary from protocol to protocol.
As has been mentioned above, it is also worthwhile looking at the amino acid composition after the mandatory GG residues at the C-terminus of the sumo tag in case there is an aa present in the protein to be cleaved that impairs digestion.
good luck!
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