GST-Fusion

[ossipow]

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