GST-Fusion

[Shahram Mori]

: >        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

My suggestion is to use another expression vector that does fusions to 
Protein A (from staphylococcus). Protein A as fusion may stop the degradation
of your protein.I think Pharmacia provides two expression vectors PRIT 5 and
PRIT2T. PRIT2T has a signal sequence that translocates the protein to the 
intracellular region away from degradative enzymes (MAYBE).
I hope it works.
Cheers
--
Shahram Mori					   _/\_
Program in Molecular Biology			  _\  /_
Dept. of chemistry and Biochemistry Box 3C	  \_  _/
NMSU  Las Cruces NM				    ||
88003