Insoluble GST-fusion

[ijiwaru at wheel.dcn.davis.ca.us]

In article <2640.199611031441 at pansy.csv.warwick.ac.uk>,
JL at DNA.BIO.WARWICK.AC.UK wrote:

> I have constructed a 64 kDa GST-fusion protein that is completely 
> insoluble.  
> 
> Curiously, when the protein is overexpressed in E. coli 
> using the T7 system, although some of the protein is insolble, some
> (about 40 %) is soluble and  fully active protein.
> 
> With the GST-system this clearly is not the case.  I have tried lowering
> growth temp. and IPTG concentration but still the GST-fusion protein
> is completely insoluble.
> 
> Has anyone else had this problem - does anyone have any ideas???
> 
> Thanks in advance
> 
> John

John,

I've had similar problems with a GST-trypsin fusion I made.  I haven't
tried this yet, but it has been suggested to me to try the expression at
28-30 degrees.  Someone suggested this saying it worked for the expression
of something else they worked with, I presume because folding of the
protein was rate limiting.  You could also try to get ahold of one of the
chaperone clones, like GroE (?), and co-expressing.  A switch to a
thioredoxin fusion partner may also work, however I do know of one
insoluble thioredoxin fusion.

Good luck,

Lyle Najita
Plant Pathology
University of California - Davis