fresh transformations for protein expression??

Michael Szardenings msz at bio.embnet.se
Thu Jan 11 07:31:14 EST 1996


In article <4ch87s$15oc at majestix.uni-muenster.de>, Torsten Boerchers
<borcher at uni-muenster.de> wrote:

> The more we were surprised that we now on two occacions
> with similar proteins encountered problems (i.e. no
> expression) which were solved by freshly transforming
> the E.coli (BL31(DE3)) with the expression vector instead
> of starting from a plate. 

Dear Torsten,
this problem is indeed very common as the many replies on the list show. I
would like to add (from my own history) a few points:

1. This is no problem that is specific for BL21, it is indeed common to
all systems where the expression of the foreign gene is not properly
regulated. In case of BL21/pET I would recommend: 
a) use media with 1-2% glucose (keeps lac-promoter down)
b) use pLysE (as others already told)
c) NEVER grow precultures or plates to the stationary growth phase, then
the cells are most sensitive to heterologous expression of toxic genes. 
d) use 30 deg C or lower for cultivation.
e) check the uninduced cultures, I once saw almost as much protein in
these as in the induced cells (return from here to 1a...)

2. Many people are claiming, that these effects are due to plasmid
instability. I have never seen any real proof for this, i.e. effects that
go beyond the normal loss of plasmids due to selection for non-expressing
clones. In the worst case I have seen growth arrest of the culture
followed some time later by rapid growing cells without plasmid, as the
ampicillin used in the experiment had been degraded during the initial
growth.
Another effect has been, that proteins, that ought to be secreted, were
much more sensitive to such effects. This may be due to the fact, that
more genes are involved in the proper expression of such proteins. I think
that spontanous mutations may result then easier in clones expressing less
protein. I have had such cases, were the plasmid from the culture was
absolutely o.k., but the expression level was x10E-4 (!) of the level
achieved under optimal conditions.

Good luck, you may need a bit
                              Michael

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