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
<[log in to unmask]> 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
>
>
>
>
>
>