GST fusion prots truncating at 27 kDa?

Daniel Harkness harkness at bio.bu.edu
Thu Jul 27 10:18:56 EST 1995


Jeff Pitman (jpitman at sdcc3.ucsd.edu) wrote:
[among other things...]

: Has anyone else seen this before, and better yet, figured out a way
: around it?

: Thanks, 

: -Jeff Pitman
:  Frustrated grad student

Dear Jeff,

I don't usually read this particular newsgroup but regardless I will give
my simple 2 cents at the expense of sounding obvious (nothing in molbio is
obvious).  When expressing my 40 kd protein as a fusion with GST I was 
getting a whole array of truncations all the way down to the 27kD GST moiety.
This was seen on a coomassie stained SDS-PAGE.  I started using a simple
protease inhib. cocktail consisting of leupeptin, DTT, PMSF and Aprotinin in my
bacterial lysis/sonication buffer and my fusion protein was purified whole 
without truncations. I was using just aprotinin before and just like voodoo,  
additional protease inhibitors helped immensely.  

If you want the working concentrations of these just email me.

Dan Harkness
Boston University
Dept. of Cell and Molecular Biology
5 Cummington St.
Boston, MA 02215
email: harkness at bio.bu.edu



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