fusion proteins - final try

Euan R. Taylor etaylor at ccu.umanitoba.ca
Wed Jun 9 19:35:55 EST 1993


Dear netters,
I am having big problems making a fusion protein construct in pmalc2 (maltose
binding protein tag, Ptac, lacQ) in E coli. All my clones are rearranged,
meaning
about 30% of the plamid/insert has been deleted by the host strain (which
is XL1-blue MRF'  previously used quite successfully for the same purposes
with a different protein in our lab.) 100% of positively hybridising
colonies have undergone this rearrangement/deletion (and no they were not
grown on IPTG). 
The manufacturers suggest that I try another vector based on a T7 promoter
rather than the LacQ which this system uses.
The system I am looking at is made by Novagen, using a t7 promoter in
series with a LacQ promoter to minimise expression leakage.
I would be interested to hear from anyone who has used this system, how
convenient is it? any problems to watch out for? Any prefernces among
people who have done a lot of this kind of fusion expression work?
Second thought, I am wondering about making the fusion construct to
express in yeast instead on the principle that what is lethal or harmful
to Ecoli may be less so to a eukaryotic host. I really want to make the
protein with a convenient tag however so I can purify it reasonable easily, I
could
engineer my own tag onto it if necessary, but I would rather avoid the
extra step. Anyone come across a
vector for yeast which is suitable for this application, which won't
rquire me to add my own fusion leader? I'm expressing (or rather not
expressing) a plant protein by the way. Anyone have any advice to offer on
the relative merits of yeast v's E.coli as an expression system for making
immunisation quantities of cloned antigen?

All advice welcome.
cheers
Euan

Usual disclaimers. These are my own opinions and probably nobody else's.
etaylor at ccu.umanitoba.ca
"All great truths begin life as blasphemies"  George Bernard Shaw 



-- 
Usual disclaimers. These are my own opinions and probably nobody else's.
etaylor at ccu.umanitoba.ca
"All great truths begin life as blasphemies"  George Bernard Shaw 



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