On 22 May 1995, Clemens Vonrhein 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 ?)
Sometimes the strains won't grow then, but a good thing that has helped
me in at least 2 cases. Try perhaps another more suitable strain for low
temperatures.
> - low IPTG concentration
In the case of the T7 RNApol systems already very little may cause very
strong induction, better to use a different expression system, that is a
more easily fine tuned system.
> - rich medium ( terific broth, ...)
On the contrary, slower growth in less rich media may be very helpful.
But there are very different opinions and experiences about this.
> - high OD and short incubation times (OD=1.0-2.0 and t=1-2h)
If the inclusion bodies form due to lack of available chaperonins, then
avoid too 'old' cultures for induction, induce and harvest latest during
linear growth.
> - adding needed cofactors (FAD, FMN, ...)
Has helped in many cases. For example the vitamin D receptor was
expressed in yeast in presence of horrible amounts of its ligand. Just to
tell you that it can be quite expensive.
> Are these rules true in general ? Is there a good and recent review ?
No!
> 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 ?
Refolding of small proteins works well, even if you want to crystallize
the protein (as you probably want to do), but large proteins, especially
when they have several domains, are very tricky. Rainer Rudolph has
written some nice articles about such problems.
> BTW, I use a pET-22/BL21 system.
Perhaps try another strain, supply the T7RNApol in this case from plasmid
pGP1-2 from Stan Tabor (Harvard Medical School), it has Kan-resistance
and a compatible p15A origin. (PNAS (1985) 82 1074-1078) I have a paper
ready for submission, where we show, that the PL-promoter controlled T7pol
gene on this plasmid can be induced at any temperature by allowing the
culture to grow long enough to get induction by the high pH forming in
the end. You may also use 'artificial' pH shifts (see also Gene 97 125-130).
OR make your own DE3 lysogen, the phage is commercially available.
Hope this helps, GOOD LUCK
Michael
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