proteolysis during expression in bacteria

Suzanne Elsasser selsasser at mediaone.net
Sat Feb 17 22:11:10 EST 2001


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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