Stable expression of very large ( >250 kDa ) proteins in human cells

Kyle Legate via methods%40net.bio.net (by legatek from hotmail.com)
Sun Jun 17 04:09:58 EST 2007


Ozan Aygun wrote:
> Dear all,
> 
> I am using the pIRESpuro vector (clontech) and trying
> to establish stables in 293 cells. Up to know, I have
> successfully established stables of proteins up to 100
> kDa by selecting as low as 1ug/ml puromycin
> concentration in 293 cells.
> 
> However, some of the proteins that I am interested in
> are above 250 and 300 kDa. I have cloned and
> transiently expressed these large clones and observed
> their expression by WB. Furthermore, when I have
> proceeded for the selections in 1 ug/ml puromycin in
> 293 cells, I have obtained several clones as usual. 
> Unfortunately, when I assay these puro resistant
> clones for the expression they seem to be either not
> expressing or expressing a shorter form of the
> protein.
> 
> Since in IRES bicistronic system theoretically a
> resistant clone should express the protein in frame, I
> have been really puzzled with this unexpected
> situtation.
> 
I don't think it has to do so much with the size of the protein you are 
trying to express, but with the identity of the protein itself. I'll 
explain why:
A while ago I was trying to obtain stable clones of a GFP-tagged enzyme, 
total size a little bigger than 100 kDa. It was cloned into an 
IRES-zeocin vector and clonal selection proceeded normally with nice 
GFP-expressing cells. However, over time (4-5 passages), the GFP signal 
was lost though the cells were still under selective pressure. I never 
characterized exactly what was going on but at the end, I had cell lines 
which were Zeo resistant but did not produce my target protein. I think 
expression of the fusion protein was somewhat toxic to the cells (it 
messed with lipid homeostasis) so they figured out a way to shut down 
its expression while maintaining the expression of the Zeo cassette. 
Perhaps the same thing is occurring to you.


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