Best protease cleavage for Fusion Proteins?

Rick Thorne rthorne at mail.newcastle.edu.au
Wed Jul 8 11:25:19 EST 1998


Dear All,

                I am planning to construct a recombinant (fusion)
molecule to be produced in CHO cells, with the ultimate aim  to produce
around 1mg of the purifed protein. Rather than producing a secreted form
of the molecule, I want to make it as a transmembrane protein (Type 1).
The complication is that "maturely" glycosylated surface expressed
molecules are required for analysis, and therefore it should not be
purified from whole cell lysates (which would be contaminated with the
precursor of the protein). The plan is to cleave it from the cell
surface by introduction of a protease site in the extracellular domain
of the protein, close to the transmembrane.

                DOes anyone know of this approach being successfully
used before. It is most common to first purify the fusion protein and
then cleave off the fusion partner (eg: GST). Therefore the type of
cleavage site is not so critical. However does anyone have a suggestion
as to which cleavage method might be most applicable to live cells??

Thanks,

Rick Thorne
Cancer Research Unit,
The University of Newcastle,
Newcastle, Australia.





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