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Hi Rob,
Thanks and I did try this already but just ended with two unhappy proteins.
This result might not be all too surprising. It might be a scenario that
the two proteins need each other for proper folding. In such a case,
when expressed individually, the proteins may aggregate in a
non-native/misfolded state into an inclusion body, and therefore, mixing
and lysing the two pellets might not work.
It might require throwing two denatured proteins and refolding together.
But in that case, who's to say that they will refold correctly. I know
refolding denatured histone protein partners works great. But I suppose
we also hear of many failed refolding sagas.
Thanks for your suggestion. Like you write, the best bet might be the
one multi-expression construct.
Raji
Rob Gruninger wrote:
Hello Raji,
Not sure if this is applicable but I will mention it anyway. I am
assuming that you are trying to obtain a complex between these five
proteins? If so I have seen people do this by overexpressing the proteins
in two different cell lines, mixing the induced cells together and lysing
them together. If you can overexpress your pET28 and pET21 constructs
seperatley perhaps this approach would work. Might be easier then trying
to get a clone containing all five proteins.
Regards
Rob
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Hi Y'all,
Non-xtallo query.
I have the foll. plasmids:
1) a pET21a vector (AmpR) expressing a single protein
2) a pET28 vector (KanR) containing a polycistronic construct that
co-expresses four proteins
While we contemplate sticking the fifth protein and making a single
multi-expression pET28 vector, I tried to co-transform the two plasmids
by bumping up the two antibiotics and that did not work.
If you have had success with co-transforming two plasmids that share the
same replication origin (for e.g., two different pET vectors) into
E.coli expression strains, could you pl. share your tricks and/or
suggestions. Do you have favourite strains/vectors when it works?
Many thanks.
Raji
--
Raji Edayathumangalam
Postdoctoral Associate
The Rockefeller University
Box 224. 1230 York Avenue
New York, NY 10021
