Hi :) In addition to excellent replies already posted, a few thoughts:
1. Do the whole-protein MS of your stuff. Assuming that you get a spectrum then a) If you are right and it is indeed your recombinant GST - you will see a mass pattern that's interpretable via analysis of probable cleavage sites. You will also glean some understanding of where the undesired cleavage (or premature termination!) occurs. b) if the suggestions of previous replies are right, then these protein(s) will be of the insect-cell origin. I have not seen this to be a huge issue in the past, however it may be due to the experience with Sf9, Sf21 cell lines and relatively low use of Hi5 in my past work. Switching a strain might help (maybe you already tried, and it still sucks, sorry). If you can't get a spectrum of the whole protein I would potentially suggest tryptic cleavage and peptide MS. You will get at least an idea whether (a) or (b) is more likely. In general (b) is tougher to fix because this means that your protein isn't being expressed in large enough amounts. You don't mention - is the insoluble fraction interesting? Is there a big fat band at the correct m.w. in the insoluble material? Perhaps a change of lysis conditions might help, or expression at lower temperature (yes, insect cells do grow at somewhat lower temperature, however they're not very happy there) or lower MOI. Also: do you *need* MultiBac system in your case? It's an excellent tool for large complexes but it's not clear from your post whether you're working with one protein or many? MultiBac has Cre/Lox leftovers in the bacmid which may be un-fun in your case for some reason (unlikely but...) Take a look at the DNA & RNA structure (predicted) in the junction region. Is there any 'funny business' with hairpins, ribozymes, unusual structures of any kind? A cryptic ribozyme can really mess up one's life (it's not likely that you have one, much more likely an early termination structure or inefficient read-through). Splicing can also be difficult to control on occasion - any presence of strong splicing sites? AG/GURAGU and YAG/RNN with a few additional parameters thrown in. This would require a fair bit of RNA to be in between the sites, too. Cheers, Artem On Fri, Mar 16, 2012 at 7:57 AM, Sebastiano Pasqualato <sebastiano.pasqual...@gmail.com> wrote: > > Dear CCP4ers, Dr. Berger, > > we have an accumulating series of GST-fusion proteins here that are all > displaying the same behavior when expressed in Hi5 insect cells with the > MultiBac system. > > What we are experiencing is a massive production of free GST and only a > limited amount of fusion protein. > > Since the linker we have engineered between GST and our protein is the one > that exists in the pGEX-6p vector series, with the preScission protease > site, I was wondering if any of you had experience of this cleavage site > being processed within these insect cells. > > Thanks in advance for the help, > ciao, > Sebastiano > > -- > Sebastiano Pasqualato, PhD > Crystallography Unit > Department of Experimental Oncology > European Institute of Oncology > IFOM-IEO Campus > via Adamello, 16 > 20139 - Milano > Italy > > tel +39 02 9437 5167 > fax +39 02 9437 5990 > > > > > >
