GST-fusion problems

Dr. Duncan Clark d_clark at nospam.demon.co.uk
Mon Mar 5 14:41:51 EST 2001


In article <980iga$22c_002 at doit.wisc.edu>, the eminent Dima Klenchin at
UW wrote
>Kevin Brady <rcxkb at unix.ccc.nottingham.ac.uk> wrote:
>:
>:Duncan, which protease inhibitor(s) would you recommend if it is
>:proteolysis, AEBSF..?
>
>Kevin, inhibitors would only help if your proteolysis 
>occurs post-lysis. If it happens during expression
>(inside bugs) - this could be both premature termination
>or true proteolysis - then there is not much you 
>can do. 

Sorry, I was initially thinking about proteolysis during expression but
should have said that the inhibitors would only work as Dima says.

For proteolysis during expression, you can try the normal tricks. Induce
at lower temps i.e. 20C, 25C or 30C rather than 37C. 

Induce for as short a period as you can get away with.

Harvest cells as fast as possible and lyse and process immediately or
freeze fast by dropping into liq. N2 or whatever.

Duncan  
-- 
The problem with being on the cutting edge is that you occasionally get 
sliced from time to time....

Duncan Clark
GeneSys Ltd.
Tel: +44(0)1252376288
FAX: +44(0)8701640382
http://www.dnamp.com
http://www.genesys.demon.co.uk






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