Rana,
I understood that these proteases are very efficient in cleavage. One time, I
had a construct MBP-3C-protein, I never able to cleave this particular
construct while I could do well with other truncations.The MBP you are seeing
might be co-purified with DHBx and unless gel band intensity suggest that its
coming from fusion protein. I feel cleavage site may not be accessible for the
protease. Might be adding few more residues might help.
Good luckSDY
Date: Sun, 4 Nov 2012 07:24:42 -0800
From: rna19792...@yahoo.com
Subject: [ccp4bb] protein cleavage
To: CCP4BB@JISCMAIL.AC.UK
Dear CCP4
I am having a problem with cleaving my fusion protein and I would be
grateful if you advice me regarding this situation, I have an MBP-DHBx fusion
protein and I am trying to cleave it using TEV protease, I have tried different
ratios and different temperatures with different incubation time but still it
will not cleave, all I observe on the gel is the bands of the fusion protein
which is 59kDa and the MBP which is 42kDa and the TEV protease which is 27kDa
and no sign of the DHBx which is 17kDa,I have also checked the sequence if
there was any problem but I could not find anything unusual the sequence was
fine ,
so if you have any suggestions regarding this situation I will be thankful
Best Regards
Rana
