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