Co-expression of two proteins in E.coli
mytelka at mendel.Berkeley.EDU
Fri Oct 4 19:48:13 EST 1996
There are at least a couple of ways to do this fairly easily: clone
both proteins onto one plasmid and clone each onto a separate plasmid
with a different (compatible) origin of replication. I've done each
of these successfully. I've also used three antibiotics at at time
without a problem (of course, each adds a bit of stress to the cells...).
Come to think of it, I think I used four once (amp, kan, tet and nal).
If you're using different plasmids, it's obviously not a good idea
to use amp on both :-) Remember that if you are going to use
BL21(DE3)pLysS, the pLysS plasmid is pACYC based, so you have to
find a third origin of replication (presumably what you currently
have is all colE1 based). And if you're using two different plasmids,
it's tough to get them both taken up in one transformation if you're
doing chemical transformation; you'll have to do it one at a time
or try electroporating (which I didn't try for this particular
application, but would probably work since it's the mammalian standard).
In article <532dtt$9g at web3.tcd.ie>, C.R.Baker <crbaker at mail.tcd.ie> wrote:
>I have two proteins, in two different amp-based expression vectors for
>E. coli. Other than transforming with the two and analysing for
>coexpression directly, is there any trick in order to get the two
>vectors into the bacteria?
>On expression one protein is pretty toxic to the cells, so is it likely
>there would be selection for those cells without this vector.
>Is the best option to clone one of the proteins into an alternative
>expression vector based on a different antibiotic e.g. kanamycin and
>select on amp-kan media? Are there any particular combinations of
>antibiotic that it is not advisable to use? Would it be going too far
>to use say BL21 (DE3) pLys S and select on amp-kan-chloramphenicol?
>I'd be grateful for any advice.
>crbaker at mail.tcd.ie
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