It is absolutely normal that the cells expressing recombinant protein slow
down in growth upon induction. You are basically making cells overproduce
the protein (that they don't need) and this consumes enormous energy and
reductant resources of the cell that would otherwise be used for maintaining
growth. In fact, the better is the expression, the slower should the cells
grow. You should not see any slow-down in the cells transformed with the
empty vector, unless it is a fusion vector that expresses another protein.
So, it is not necessarily due to toxicity of your protein that the cell
growth slows down. You don't see decrease in cell number (OD), right? So
there is most probably no dying, and you cannot use the term "toxicity"
then. Toxicity is when you start induction with say 1 gram of cells and end
up with 0.5 grams of slimy mass instead of 'healthy' looking cell pellets.
Try starting induction at higher OD (0.75-1.0) and induce for a short time
(1 h). If that works, try optimizing induction time - you might get more
protein with 2-3h inductions. Sometimes the products of metabolism
accumulated in denser cultures can adversely affect expression. In such a
case you could wash and resuspend the cells in a fresh medium shortly before
induction (you can play with cell concentration here, too). If you start
getting inclusion bodies, try reducing induction time and/or temperature
(20-30C), cell concentration. However, sometimes inclusion bodies are
better, but you will have to work out refolding conditions.
Emir
"Bertrand Collet" <b.collet at abdn.ac.uk> wrote in message
news:3D771550.C6F97A16 at abdn.ac.uk...
> 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
>>
