coulier at infobiogen.fr
Sat Oct 5 01:48:10 EST 1996
On Fri, 4 Oct 1996, Tim Angelotti wrote:
> I am trying to clone a cDNA encoding a extracellular protein. However, I
> noticed that all of my positive clones (correct orientation) grew very
> slowly and thus gave poor yields. I am using Bluescript. I suspect that
> this sequence is toxic, and talking with a few others working with this
> gene confirmed it may be. Now the question, what is toxic about it? Is
> it the sequence of nucleotides, or is it producing some of the protein
> accidently and this is toxic, or is it most likely an alternate open
> reading frame coming from another cryptic promoter? I have heard all
> three and was wondering if anyone could shed light on this. If it is any
> of the above, does this mean that any DNA I recover is most likely
> screwed up, rearranged, and would using ABLE or TOPP cells from
> Stratagene help?
> Tim Angelotti
> TU Muenchen
> Institute for Pharmakologie
I once got a similar problem trying to express a growth factor in e.coli.
Using 35Smethionine pulse label, I figure out I had a very low expression
within 15 min after induction, and then nothing.
Removing the signal peptide solved my problem.
Hope this help.
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email: coulier at infobiogen.fr
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