Refolding a GST-fusion-protein --- Help needed!

Wulf Blankenfeldt wub at gbf-braunschweig.de
Tue Sep 26 04:20:28 EST 1995


Dear all,

we are currently working on a GST-fusion-protein using the pGEX-2T
vector from Pharmacia. Expression in DH5alpha is quite high, but
unfortunately the majority of the protein (more than 90%) goes in
inclusion bodies. Changing growing/induction conditions (lowering
temperature, IPTG concentration etc.) did not help, so we decided to
work with the inclusion bodies, prepurifying the protein by washing,
solubilizing it in buffered urea supplied with DTT and EDTA in order
to then start a renaturation procedure (dialysis or dilution).
To our further misfortune the fused protein, which is membrane
associated and has one Cys-Cys bridge in about 200 residues, does not
possess any yet know enzymatic activity so we are currently monitoring
the refolding by use of the CDNB-assay, assuming that if the two
Cys-Cys-bridges in GST are built correctly the one in the fused
protein will be, too.

Our questions:

1.) 	
Has anyone ever tried to refold a GST-fusion-protein before and is
willing to share his/her experiences?

2.) 	
In order to reshuffle disulfide bridges one usually uses redox systems
containing reduced (GSH) and  oxidized glutathione (GSSG). As
glutathione is the substrate for GST in the CDNB-assay, how can one
find out the best conditions for refolding concerning the GSH/GSSG
ratio? Should one dialyze the samples and then determine protein
concentration prior to the kinetics assay (sounds quite a trouble and
not very promising)?

Any help/suggestions would be appreciated!


Thanks in advance,


			Wulf




Wulf Blankenfeldt

Gesellschaft fuer Biotechnologische Forschung
MSF
Mascheroder Weg 1
38124 Braunschweig

Germany

 phone: Germany (0)531-6181 372
   FAX: Germany (0)531-6181 355
e-mail: wub at gbf-braunschweig.de





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