GST-Fusion

[I-Jen Huang]

In article <iert237-030395125450 at jah10.oac.tju.edu> iert237 at tjuvm.tju.edu (zac ) writes:
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>Newsgroups: bionet.molbio.methds-reagnts
>Subject: GST-Fusion
>Message-ID: <iert237-030395125450 at jah10.oac.tju.edu>
>From: iert237 at tjuvm.tju.edu (zac )
>Date: 3 Mar 1995 17:58:52 GMT
>Followup-To: bionet.molbio.methds-reagnts
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>Organization: Thomas Jefferson University
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>        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

Have you tried lower the growing and expression temperature?  We had a 
similar problem before until we changed the temperature and included 100 mM 
EDTA in the lysis soultion.  The 100 mM EDTA will not effect the binding of 
GST to beads.

I-Jen Huang
Grad Student
USU
GR8BN at CC.USU.EDU