GST-Fusion

R. Rex Denton DENTON at biomed.med.yale.edu
Sat Mar 4 17:00:23 EST 1995


In <iert237-030395125450 at jah10.oac.tju.edu> iert237 at tjuvm.tju.edu writes:

> 	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


Hi,
Someone in our large group has reported some problems similar to those that
you are presently encountering.  If you are using routine hosts for your
expression (ie DH5alpha, JM109 etc.), we would emphatically suggest changing
your host system to one that has the genotype OmpT.  These host are defective
for a specific kind of protease within the E.Coli, that cleaves outermembrane
proteins.  BL-21 is such a host, and comes wioth the Pharmacia expression
vector pGex.  This host is also EXTREMELY PRIMITIVE and HARD TO TRANSFORM
as well not mutated for RecA (I believe). Therefore, it is recommended 
that most vector manipulations, subcloning, be performed in other more
hospitable hosts, and this one be used only for expression of your product.  
Best of Luck,
R.Rex Denton
Yale
My opinions, etc. not anyone else's
 



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