Histag toxicity in E.Coli
Frank O. Fackelmayer
fof1 at chclu.chemie.uni-konstanz.de
Fri Feb 19 11:19:22 EST 1999
In your question there are two things to comment on:
1. Protein size: E.coli tends to rapidly degrade small proteins. As a
rule of thumb 10kD is the lower limit. You can be lucky with your 9kD
protein, but I guess you´ll get stability problems. Two strategies are
often used to overcome this problem. You can either use a quite big
fusion partner (e.g. GST or thioredoxin; there are vectors that encode a
protease site so you can later cleave off your peptide), or secrete the
protein into the medium. In my lab, we routinely use this second
strategy, employing the pEZZ18 vector from Pharmacia (no affil.). Works
like a charm even for a 5.2kD peptide.
2. checking toxicity: Despite the fact that some proteins are REALLY
toxic to E.coli, in many cases "toxic" is an euphemism for "didn´t
work". Anyway, your strategy to assess toxicity is unlikely to work,
because the high-level expression of even non-toxic proteins often
result in a stop of growth. The bugs are not dead, but simply too
exhausted to grow. There are staining methods to tell dead bacteria from
live ones, but I guess you´re more interested in getting hands on your
protein than playing around with such stuff.
Hope this helps,
Ian Mc wrote:
> We have produced a histag construct of a small piece of our protein (9kDa)
> but it does not seem to express at all. I have heard that sometimes the
> protein can be toxic to the E.Coli. So I wondering if we could use plates
> +/- IPTG to check if the bugs will grow or not.
> Is this feasible? Has any one else tried this before? Or have a better method?
> Ian Mc
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