proteolysis during expression in bacteria

Randy rldurren at bchserver.bch.ncsu.edu
Tue Feb 20 13:23:42 EST 2001


Suzanne,
I had similar problems. What I did was to add three different inhibitors to
the buffer used for the French Press:

PMSF (in 2-propanol) 100ug/ml
TLCK (in Na Acetate) 50ug/ml
TPCK (in ETOH) 100ug/ml

This was used for the crude extract from the French Press and the extract
applied to a His-Bind column. The purified protein is then applied to a
DEAE-Sepharose column. In this buffer I just use 1mM EDTA and 1mM EGTA,
pH8.0. These steps solved my problem with proteolysis. If you use a His-bind
column, DO NOT use EDTA/EGTA, it turns the matrix an ugly orange color.
I hope this is a help for you.

Randy Durren
Research Assistant/Lab Supervisor
rldurren at unity.ncsu.edu
Voice: 919-515-5806
Fax: 919-515-2047


"Suzanne Elsasser" <selsasser at mediaone.net> wrote in message
news:3A8F3D3A.9D4C5D14 at mediaone.net...
> Greetings All,
>
> Currently, I am expressing a number of GST fusion proteins
> and am experiencing proteolysis problems.  Upon
> purification, I obtain mostly truncated forms of a few of my
> fusion proteins. (The others look great.)  These proteins
> are being expressed in BL21(DE3) cells, the vector backbone
> is pGEX-2TK, expression is induced at 25 degrees, and the
> extracts are prepared in the presence of protease inhibitors
> (aprotinin, pepstatin, AEBSF and EDTA) by French press
> lysis.  I suspect that the lysis occurs in vivo, and am
> curious about whether there are bacterial expression strains
> engineered to contain fewer proteases than BL21(DE3) cells.
> I would be happy to hear from anyone who has successfully
> troubleshot this particular problem.
>
> Thanks a bunch,
> Suzanne
> _____________________________________________________________
>
> Suzanne Elsasser
> Home: 617-354-2069, selsasser at mediaone.net
> Lab: 617-432-1519, selsasser at hms.harvard.edu
>
> "There are four things that we want from ourselves and the
> people in our lives:  passion, compassion, humor, and
> style."
>
>







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