Toxic protein for E. coli (M15)

Frank O. Fackelmayer Frank at
Fri Sep 6 05:43:04 EST 2002

Hi Bertrand,

In addition to what the others said, the following hints may be helpful

* it is quite common that you don´t see expressed fusion protein in the
soluble fraction. In fact, many proteins expressed in E.coli end up in
inclusion bodies with not even a trace of the protein in soluble form.
To check _total_ expression, we routinely test whole bacterial extract:
1ml of induced culture, spun down, resuspended in 100ul of SDS gel
sample buffer, sonicated to reduce viscosity (important!), and 15ul per
lane analysed by SDS PAGE.

* playing around with temperatures and IPTG concentration never did the
job for me, nor for anyone else I know. It is often recommended here and
everywhere, but noone was ever able to provide convincing evidence that
it actually works. In fact, IPTG either induces or not; the expression
level cannot be regulated. For us, 0.2 mM IPTG is sufficient to give
full induction, and 0.1 mM does not induce at all. The actual effective
level of IPTG may depend on the strain you use, of course.
Changing temperature down to 28° is also often recommended. For us, this
did not change anything. To get results, the temperature must be lowered
to below 18° to really change the bacterial metabolism in a way that
_may_ affect expression levels and solubility of a protein. But still,
this is guarantee that it works. In the two cases where I tried it, the
protein simply was not expressed any more...

* coexpression of chaperones is recommended by some people. We tried all
of them. It didn´t work for any of our proteins. Of course it may be
different for other proteins, any we were just out of luck...

* the same is true for growing the bacteria under stress conditions
(which will turn on endogeneous chaperones), eg. in LB with 6% EtOH. ->
no way to improve solubility.

* The formation of inclusion bodies does not seem to be dependent on the
expression level: we have proteins that are expressed poorly (no
additional band detectable in coomassie stained gels) and still form
inclusion bodies. So, reducing the expression level may give you lower
yield of still insoluble protein.

* sometimes the induction time is too long. We did not observe a protein
we wanted after 4h of induction, but it was there perfectly after 30min.
A time course showed that the protein started to be visible after 10min
of induction, increased up to 40min, and then decreased again (most
probably because it was degraded). We finally purified that one after
30min of induction.

So, what lession did we have to learn from that?

1. When we need lots of protein and don´t care about the activity
(usually for producing antibodies), we are happy to get our protein in
inclusion bodies: it is easier to purify, usually is cleaner than
protein purified from the soluble fraction, and the yield is often
amazingly high.
2. When we need soluble protein, changing the expression system must be
considered. The high number of expression systems on the market shows
that that there is not _the one_ that works in most cases.
Unfortunately, there is no general rule which one works best for a
particular case...

For small proteins (up to, say, 40kDa), secretion into the medium was
quite good for us (pEZZ18 vector from Pharmacia was used here).
In one case, simply changing the vector to one containing a lacIq gene
worked for us (pRSET -> inclusion bodies; changed to pET -> soluble).
The expression of fusion proteins with a highly soluble tag such as
thioredoxin has been reported to work. We haven´t checked it, so I
cannot comment on it. The same applies for intein fusion proteins.

What about expression in a eukaryotic system? It may be a "simple"
eukaryote such as Pichia, an insect system (such as the systems based on
baculovirus), or even mammalian systems. For us, proteins totally
insoluble in E.coli were perfectly soluble and active when expressed in
human cells. Of course, the yield is way lower than a working
overexpression in bacteria, but for many applications it is perfectly
sufficient (e.g. band shift assays for DNA binding proteins, or
enzymatic assays).

hope this helps,

Bertrand Collet wrote:
> Dear all,
> I am new to recombinant protein production and I am attempting to
> produce a fish DNA-binding protein in E. coli.
> I use the pQE30 expression vector in M15 bacterial strain.
> I noticed that the growth rate of the bacterial cultures is the same
> without inducer for pQE30 only and for pQE30 expressing my protein.
> However, when I induce with IPTG (1mM) pQE30 continue to grow normally
> but my expression plasmid culture grow very very slowly: It appears that
> my protein is toxic for E. coli but that I do not have any leaking
> expression.
> Under these conditions, is there a good method to make E. coli produce a
> toxic protein, or do I have to switch to
> another expression system ?
> I tried to reduce the level of expression (lower temperature 28 deg,
> lower IPTG concentrations ... but no clear results so far).
> Thanks in advance for any advises,
> Bertrand Collet

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