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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 > *** For details on how to be removed from this list visit the *** > *** CCP4 home page http://www.ccp4.ac.uk *** > > > 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 > > -- Robert J.Gruninger B.Sc. Graduate student Department of Chemistry & Biochemistry University of Lethbridge, 4401 University Dr. Lethbridge, AB, Canada, T1K 3M4 Phone:(403)329-2746 Fax:(403)329-2057
