Clemens Vonrhein email vonrhein at bio5.chemie.uni-freiburg.de wrote:
>The subject says it all: I want to avoid the expression of a protein
>as inclusion bodies.
>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, ...)
When I was doing my PhD I did some protein expression work, I was using pET11/
BL21. In my case I found that I needed to induce the cells before the OD
reached
about 0.5 in order to produce mostly soluble protein. Inducing later, although
giving a similar level of protein on a gel, gave a much smaller peak on my
first
column. Although I didn't show definitively that inclusion bodies were being
formed it suggests that late induction may not necessarily be the answer. I
think it is difficult to come up with hard and fast rules for this sort of
thing, there are so many variables.
Good luck!
Rob
--------------------------------------------------------------------------------
Rob Brooksbank The Sanger Centre,
Hinxton Hall
email: rab at sanger.ac.uk Hinxton
phone: (01223) 494949 Cambridge
fax: (01223) 494919 CB10 1RQ