In article <3pqbq5$lu1 at n.ruf.uni-freiburg.de> vonrhein at bio5.chemie.uni-freiburg.de (Clemens Vonrhein) writes:
>The subject says it all: I want to avoid the expression of a protein
>as inclusion bodies.
My experience has been that once you have inclusion bodies, you're faced
with a lot of work to get it soluble and active, and with low probability
of success. I would suggest another project (really). I don't think
many (or maybe any) proteins that have been refolded have crystallised
for instance (some indication of their lack of homogeneity). I have
tried different clones (chopping off a few residues at the ends) which
succeeded in making a soluble expression system, with a protein of
40kd, but no success with a bigger protein. Changing from e.coli to
yeast fixed one system I know of.
Joe Mack
mack at ncifcrf.gov
>>As far as I read the literature, there are some rules of thumb:
>>- lower temperature (30, 25, 20, 15 degrees Celsius ?)
>- low IPTG concentration
>- rich medium ( terific broth, ...)
>- high OD and short incubation times (OD=1.0-2.0 and t=1-2h)
>- adding needed cofactors (FAD, FMN, ...)
>>Are these rules true in general ? Is there a good and recent review ?
>What are your own experiences ?
>>Is it worth trying to get soluble protein ? Or is it better to get a
>lot of protein as inclusion bodies and try to refold it ?
>>BTW, I use a pET-22/BL21 system.
>>Any hints/answers/tips .. highly apreciated.
>>Thanks
>>Clemens
>---
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>* Clemens Vonrhein email vonrhein at bio5.chemie.uni-freiburg.de *
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