Help with overexpression in Leishmania

[ijpinto from ibmc.up.pt]

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