Expression in Pharmacia pGEX-Vectors
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 ?
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 Krasel, U Wuerzburg, Dept. of Pharmacology, Versbacher Str. 9 */
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