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

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.

Here are my questions regarding this confusing issue:

1. What can I do best in this situation to get an
expressing clone? I.e: do you think increasing the
puro concentration might help? or should I select,
propagate and screen more and more colonies? or Should
I give up this and try a completely different
approach?

I would be grateful if you can give me any suggestions
with stables of very large proteins in IRES system or
with any other methodology. Since we do not have any
experience with large proteins in this system, I am
continuously wondering whether obtaining a stable
clone is still possible with such large proteins.

2. Is there any theoretical basis or explanation for
this "leaky" expression of the puro resistance gene
independent of the upstream ciston?

All of your comments and suggestions will be greatly
appreciated.

Regards,



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