GST-Fusion

[L Bruce Sk]

>In article <iert237-030395125450 at jah10.oac.tju.edu>,
iert237 at tjuvm.tju.edu (zac ) says:
>
>        We are working with the GST-Fusion system and are having an
extremely
>difficult time getting a fusion product that is not degraded.  We have
>looked at almost every parameter, including induction time, culture
volume,
>sonication/lysis conditions, you name it.  We were also told some
>constructs just do no work well do to unknown problems linked with the
size
>of the protein we have cloned in.  We have now made a new construct that
is
>totally different in size (much larger but encoding the same initial
>region) and have found that degredation is so bad that we barely have any
>fusion product.  If anyone can help, I would GREATLY appreciate it. 
-Note:
>we are using the pGex-2T vector
>
>Darren Boehning
>Thomas Jefferson University
>boehnin1 at jeflin.tju.edu
>I have had such problems too, and I found an easy way to have less of
>degradation 
>products by growing the cells in LB medium WITHOUT GLUCOSE, for only
three
>hours after induction with IPTG, if necessary you can also try to grow
the cells at
>30c after the induction (that with very usefull in my hands); finally I
would strongly
>recommand you to switch of E. coli strain to try a Omp minus.
>Good luck


Pharmacia can supply you with a 'GST Gene Fusion System' Troubleshooting
and Procedural Guide, now in its second edition for no charge.  Contact
your Pharmacia rep or the company directly to obtain (800) 526-3593