Expression in Pharmacia pGEX-Vectors

Cornelius Krasel krasel at wpxx02.toxi.uni-wuerzburg.de
Tue Sep 8 09:55:57 EST 1998


In bionet.molbio.methds-reagnts Lars Komorowski <larskomo at physik.mu-luebeck.de> wrote:
> Who has protocols or can deliver me some experienced problems ?

Possible problems:

1) Protein doesn't express.

2) Protein expresses in inclusion bodies.

3) Protein expresses, but is proteolytically cleaved.

Possible solutions to all problems:

a) Lower the temperature (some proteins are better expressed or fold more
   stably at lower temperatures) from 37°C to 30°C, 25°C, 20°C.
b) Play around with induction times and IPTG concentrations.
c) Try switching hosts (e.g. BL21 instead of JM109).
d) If protein is cleaved during the purification process, try different
   lysis protocols, possibly include protease inhibitors into your
   lysis buffer etc.

(I have no experience yet with coexpression of chaperones.)

--Cornelius.

-- 
/* Cornelius Krasel, U Wuerzburg, Dept. of Pharmacology, Versbacher Str. 9 */
/* D-97078 Wuerzburg, Germany   email: phak004 at rzbox.uni-wuerzburg.de  SP4 */
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