overexpressed protein sometime gives two bands
ash_mountain at yahoo.com
Thu Jan 24 10:04:27 EST 2002
I can add that for some small proteins I see a mobility difference due
to oxidation of the protein. If you normally add DTT or BME to your
solutions this is probably not the problem. Note that
post-translational modifications such as phosphorylation cannot be the
problem here as you are expressing in E. coli!! Proteolysis seems
quite feasible provided you see the anomalous band migrating faster
than the expected product. If the latter is not the case, it is more
likely to be a contaminant. Do you add PMSF and/or other protease
inhibitors during purification, especially during cell lysis?
what you can do as a control, if a facility is available, is cut out
the protein bands or blot them and have them N-terminal sequenced or
mass-spec'd (as suggested by Artem). Of course, using an antibody
would be even easier. This will tell you whether both bands originate
from the same protein (hence, proteolysis could have occurred) or
whether you are dealing with a contaminant. If the latter is the case,
you should consider optimising your purification procedures. The fact
that you do not always see the two bands is a bit suspicious; it could
suggest that maybe you are not consistent in your procedures, or you
do not use fresh solutions each time for purification, or perhaps your
protein is very sensitive to small variations in environment.
Do you normally cleave the his-tag or leave it on? If you cleave it,
you could compare cleaved and uncleaved protein on the same gel to see
if there is a difference in mobility. If so, inefficient cleavage is
probably the problem.
There's lots more suggestions to be made, too many to list here, but I
hope these are helpful.
"Artem Evdokimov" <AEVDOKIMOZ at cinci.rr.com> wrote in message news:<BV518.16689$yy3.2263020 at typhoon.neo.rr.com>...
> Dear Tina,
> You can't know for sure until you determine accurate molecular weight of the
> two sub-species. The easiest and the most reliable way to do so would be to
> submit them for MS. If your difference is really that large (5 kDa is
> enormous!) then you will definitely see it clearly and probably will be able
> to determine what's going on. Reasons for a 5 kDa difference can be as
> simple as proteolysis and as complicated as shock-induced co-expression of
> various bacterial proteins, rare codons, and protein modifications which can
> give rise to an 'apparent' m.w. difference without actually casuing a 5 kDa
> real weight change. A classical example of the latter would be
> phosphorylation since SDS-PAGE of a mixture of phosphorylated and
> nonphosphorylated protein will often (almost always, in fact) run as two
> bands due to charge difference.
> "user" <q6252397 at fernuni-hagen.de> wrote in message
> news:a225sc$nf2$1 at f1node01.rhrz.uni-bonn.de...
> > Dear all,
> > I'm overexpressing different proteins (phage, E. cloacae and C. freundii,
> > with and without his-Tag) in E. coli.
> > Usually I have a good expression and a single band during purification.
> > I wonder why I sometimes get two band, which are there all the time during
> > purification. They differ only very little in Mw. Probably much less than
> > kD.
> > I suppose, it is the same protein because it seems to bind on the column
> > exactly like the "one-band-protein".
> > Any explanations for this?
> > TIA,
> > Tina
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