In article <4a7v5g$7kq at news.cais.com>, rover at ns1.livnet.com (Rover) wrote:
> A few weeks back I asked a question about isolating the receptor using
> the ligand and received some very informative replies and email for
> which I am very thankful.
> I comes down to the two hybrid system, or luckily for us, since we
> have a secretory gene, the panning technique. From what we can gather
>> 1) Has anyone with a secretory gene tried both techniques and can
> offer an opinion?
> 3) Are there any other pitfalls to the two techniques that aren't
> mentioned in the literature that should be taken into consideration.
> --Rover The Mad Molecular Biologist
>rover at livnet.com>barlow at picard.evms.edu
I'm not speaking from experience here, but only of basic cell biology and
logic. If you are working with a protein that goes through the secretory
pathway (i.e. ER, golgi, and on...) how are you going to get your fusion
protein that has a DNA binding domain into the nucleus? If you remove the
signal sequence or place the DNA binding part of the fusion first, how do
you know that your protein will fold properly. It will need the oxidizing
environment of the ER to form disulfides (if it has them), and other
lumenal chaperones may be essential for the proper folding of your
receptor. So, IMHO, the two hybrid system has an extremely slim chance of
working. Somebody please correct me if someone has figured out how to get
around this basic fact of protein trafficking.
John A. Newitt, Ph.D. | <newitt at nih.gov>
National Institutes of Health | FAX: 301-402-0387
Bethesda, Maryland USA |