Dear all,
Can anyone help a colleague of mine who's having a serious problem with
over-expression of membrane proteins in Leishmania? Thanks a lot!
Jorge
Dear all,
I work with membrane proteins of Leishmania and I have been having some
problems.
First, does anyone have experience with chimeras? I did a GFP and a c-cmyc
fusion protein and none of them could be detected by western blot. Both
fusions are C-terminal, though I also did two N-terminal c-myc tagged
proteins and again nothing was detected.
Second, I tried to produce antibodies against those membrane proteins
using recombinant polypeptide and although it recognizes the recombinant
truncated protein, it does not recognize in parasite total extracts (in
SDS-PAGE). Is this common?
Due to this result I introduced an episomic copy of the gene in order to
over-express the proteins in the parasites. Since I could not detect the
proteins by western blot, I went back to qPCR trying to understand if
there were at least higher levels of mRNA. Indeed I saw a fold increase of
around 70 for one of the proteins and 4 for another one. The other 2
proteins did not show any increase in the mRNA levels.
So, in conclusion, I wonder if the parasites could be able to eliminate
the mRNA so fast that no protein is detected and how come the parasites
don’t have any mRNA if they carry an episomic copy of the gene inserted in
an expression vector.
I know this is a lot, but can you help me?
I thank you all in advance,
Tânia Cruz
Iron Genes and Immune System
Institute for Molecular and Cell Biology
Rua do Campo Alegre, 823
4150-180 Porto
Portugal
Phone: +351-226074956
email: tcruz from ibmc.up.pt
