Dear Rana, I think you need to clear up some confusion about this experiment. MBP fusions suffer from a number of drawbacks depending on what you are doing. First, did you use the MPB domain to purify the fusion protein (with an amylose column)? If so, you also purified native MBP from the E. coli as well (and there is a good amount in the periplasm). Therefore you should expect to see MBP before and after TEV cleavage, regardless of whether you have a fusion or not. Second, did you see the MBP fusion on SDS PAGE, particularly on Westerns with anti-MBP? Depending on your answers, we can troubleshoot your situation.
The MBP fusion vectors we have made incorporate an N-terminal His tag, followed by MBP and TEV, so we can purify the fusion either by Ni-chelation or amylose column chromatography (or both). Also we have experienced cases where, despite our best efforts, MBP fusion either is a truncated expression fragment (mostly MBP) or has a relatively inaccessible TEV site. For example, does DHBx dimerize? This could block access to the TEV site. But first, does the MBP-DHBx fusion exist and did you purify the fusion with an amylose column? Cheers, Michael **************************************************************** R. Michael Garavito, Ph.D. Professor of Biochemistry & Molecular Biology 603 Wilson Rd., Rm. 513 Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334 Email: rmgarav...@gmail.com **************************************************************** On Nov 4, 2012, at 10:24 AM, rana ibd wrote: > 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
