fresh transformations for protein expression??

Andy Phillips andy.phillips at bbsrc.ac.uk
Fri Jan 5 09:10:10 EST 1996


krasel at wpxx02.toxi.uni-wuerzburg.de (Cornelius Krasel) wrote:
>In my and my colleagues' hands, the expression level in different
>clones of transfored BL21(DE3)pLysS can be quite different. Therefore
>we transform them, screen for clones which express the protein in
>high amounts and make a glycerol stock of this clone.
>
>It has happened once to a colleague of mine that he had to make
>a new glycerol stock because the old one didn't express sufficient
>amounts of protein any more.
>
>I have no idea why this might be the case. Maybe the levels of
>pLysS are different in different clones?

This phenomenon isn't restricted to strains with pLysS. In a previous life, I 
expressed ribulose bisphosphate carboxylase (Rubisco) genes in JM83, and found that 
levels of enzyme activity varied enormously between individual isolates. Kept as a 
glycerol at -20oC, the level of expression we could get when using this to 
inoculate a fermenter declined over a period of months. We then regenerated a 
fresh, highly-expressing stock by re-transforming JM83. At the time, we speculated 
that the cause of this could be expression of the E.coli chaperonins, necessary for 
assembly of the Rubisco holoenzyme.

Andy
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