I am in search of some advice.
i have been trying to (over)express a 70kDa transmembrane (12 putative)
transporter glycoprotein. Although transient mammalian cell transfection
is carried out in the lab expression levels are quite low. I have tried
E. coli expression, as a GST fusion and induction proves toxic to the
cells. Material recoved from an affinity column ranges from 30-75kDa
and using an antiGST antibody on whole E.coli boiled in sample buffer
I see this truncation (proteolysis or premature translation
termination?) occurs in vivo. The yield can be estimated as approx
40-50ug/litre full length product (short induction times/room temp etc.)
But to cut a long story short I also have tried a tac promotor-hexahis
vector, T7 based hexahis vector and a couple of pET vectors with
much the same story.
In parallel I have also tried expression in Pichia - made the pHIL-D2
construct, confirmed the cDNA is there by southern analysis but have yet
to see protein expression. At present I am carrying out some northerns.
I was wondering if anyone could recommend another yeast expression
system. Would episomal plasmid based expression maybe in Saccharomyces
be worth trying, which promotor? under constitutive or inducible
I would be most grateful for help, information or advice on such
systems and experiences with them.
Thanks for your time.
Cara Baker (crbaker at mail.tcd.ie)
Dept. of Biochemistry
Trinity College Dublin