pRSET expression system troubles

Joe Bagshaw jbagshaw at wpi.edu
Sun Aug 25 18:33:41 EST 2002


Hello again,

Thanks to everyone who has replyed so far.  I am currently inoculating
cultures and growing them at room temp to see if this makes a difference.
Here is a little background for those that requested it.  The gene is in
pRSET A and has already been sequenced so I know it is correct.  I am using
BL21:DE3 pLys for expression.  The insert codes for a protein called PG-1.
This protein is bacteriacidal but it functions by coming together in groups
and forming unregulated pores.  I am thinking that even though it is toxic
the massive his/x-press tag complex will hinder its activity.  The expected
fusion protein should be 5-6 kd in size.  My inductions so far have been
with 1mM IPTG when the OD 600 was between .4 and .6 after growth at 37
degrees.  the cultures remained at 37 degrees after induction.  Hope this
info helps some.

Alex Borrelli

"Joe Bagshaw" <jbagshaw at wpi.edu> wrote in message
news:ajrb0v$2ic0$1 at bigboote.WPI.EDU...
> Hello all,
>
> I was wondering if anyone here has any experience expressing proteins
using
> Invitrogen's pRSET system.  I am attempting to use it but so far it does
not
> appear that my proteins are being manufactured.  If anybody else has
> experienced troubles similar to this with this system and could give me
some
> suggestions about how to fix them I would be very greatful.
>
> Thanks,
> Alex Borrelli
>
>





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