Strange size of pET-22b expression product

EK someone at microsoft.net
Thu Feb 26 12:51:38 EST 2004


"Mikael Niku" <mikael.niku at nospam.vetmed.fi> wrote in message
news:c1kond$88o$1 at oravannahka.helsinki.fi...
> > periplasmic prep. I suspect the periplasmic prep will contain both
> processed
> > and unprocessed protein species and will show 2 bands on the SDS-PAGE,
>
> This is in actually the case with an extracellular construct: two bands in
> the periplasm, one (smaller) in medium.

So it gives you an idea of what apparent MW those proteins run at on your
gels. Don't expect the difference in *apparent* MW between your proteins be
necessarily corresponding to MW of the markers. These markers are different
proteins, and mobility does depend on aminoacid composition of the
particular protein.


> With the cytoplasmic construct, I only see one band (I guess this is
> somewhat logical, see below).
>
> > By the way, do you see a lot of protein excreted into the media?
>
> With the cytoplasmic construct, it's mostly in the medium. A significant
> amount is also in the periplasm. But in fact I don't see much at all in
> inclusion bodies (the extracellular constructs do more of this).
>
You haven't told us before that you were using 2 different constructs,
extracellular and cytoplasmic. If I understand you correctly, and, unlike
extracellular, the cytoplasmic construct does not have PelB leader sequence,
the fact of having most of recombinant protein expressed from the
intracellular expression vector found in the media indicates that your
protein causes cell lysis. Otherwise, I cannot find other explanation why it
ends up in the media.

-Emir





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