protein expression problem
Frank O. Fackelmayer
Frank at Fackelmayer.de
Tue May 29 05:07:07 EST 2001
Ricky LEUNG wrote:
> Hi everyone,
> I have some difficulties to express my protein in COS-7 cells.
> I have cloned the insert into pCDNA3.1 vector and checked the reading
> frame by sequencing. Then I transfected it into COS-7 cells by Lipofectamine
> Plus and performed stable transfection with 750 ug/ml G418
> selection. After 4 to 5 weeks, I harvested the cells
the main problem is usually that only a fraction of the stably
transfected cells produce your protein (so you wouldn´t see it by
western blotting of extracts from the whole population). This is because
the expression vector often integrates in a way that the gene of
interest is destroyed or inactivated. This is particularly a problem
when the gene is quite big in relation to the rest of the vector (or the
gene negatively affects cell proliferation). Thus, the cells may become
resistant to G418 but do not produce your protein. The only way around
this is to select for single cell clones that actually make your
protein. This is not difficult but lots of work. You have to dilute the
cells when splitting so to deliver single cells into the wells of a 96
well dish, let them grow, and analyse all clones that grow for the
presence and quantity of your protein. Then, expand these cells
(optionally repeat the "cloning" of individual cells to make sure the
line is monoclonal), and go ahead.
> and did the western
> blot using Anti-His Ab from Santa Cruz. No band was detected.
but you DO have a positive control, right?
> On the other
> hand, I extracted total RNA from the transfected COS-7 cells and did the
> RT-PCR. The insert was over-amplified when compared with the control
> vector without insert.
RT-PCR will detect the RNA of those cells that actually make the RNA
(and the protein).
> So, what is the problem? No protein was found. Is
> it related to translation problem?
maybe, but this is not as usual as the problem described above. It may
occur e.g. when the codon usage is very different from that of the host
cells (e.g. bacterial genes in eukaryotes or vice versa), especially
when there are rare codons close to the amino-terminus.
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