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Dear Raji,
If you have trouble with creating the co-expression constructs, then you may
want to know about 3 co-expression plasmids we developed at my last position
at The University of Georgia. They are Gateway cloning compatible (ligation
independent) and can be used with the traditional Gateway plasmids giving
you up to 4 plasmids per cell. You can get them from the Wang lab at UGA.
The plasmids use Gateway cloning as I mentioned and compatible individual
origins of repication. In order to overcome the huge N-terminal tags we used
to add a Tobacco Etch Virus protease site (or your choice) in the 5' primers
to cut the N-terminal tags.
As an added benefit this same system allows you to clone each protein into
up to 4 plasmids at the same time, in the same tube. You tranform the LR
clonase reaction (containing the mix) and chose for the individual
consrtucts with different antiobiotic seletion plates. This allows you to
have each of your genes in 4 differnet plasmids so you can mix and match the
plasmids/genes to help your expression complex formation screening. Also
there are at least fifty of the regular Gateway plasmids floating around out
there with many fusion protein or promoters. If you are interested, let me
know I can point you to them.
Hope this helps
Peter
----- Original Message -----
From: "Raji Edayathumangalam" <[EMAIL PROTECTED]>
To: "Rob Gruninger" <[EMAIL PROTECTED]>
Cc: <[email protected]>
Sent: Sunday, July 09, 2006 9:34 PM
Subject: Re: [ccp4bb]: Co-transforming two different plasmids
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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
