expression problems in mammalian cells - (Nov/22/2006 )

Hi everybody,

I have been experienced several problems for the last months with the recombinant expression of one protein in mammalian cells.

The protein (20 Kda) has a signal peptide and an His-Tag at the N-terminus. When I transfect my mammalian cells (Jurkat), and after selecting transfected clones using appropriate antibiotic, I test the supernatant using an anti-His antibody (by ELISA or by Western blotting) and I see noting at all.
On the contrary, the transfected-cell control (using the same plasmid with eGFP) expresses very well the eGFP.

Maybe is the signal peptide and I have problems with the secretion of my protein...I verfied the sequence and it is ok.

Has anybody experienced something similar? In that case, how did you proceed?

Thank you for any help!!!

We had that problem and I ended removing the signal peptide. Since then overexpression is good and protein is not secreted

Did u check if everything is in frame in ur construct?

It's a good idea to test your plasmid for expression capabilities prior to selecting.
If you transfect your construct into cells (especially ones easily transfected such as attached cell lines, Jurakts have lower efficency rate) you should be able to collect the proteins from these cells 24-48 hours later, do a Western and see expression. If you do not see expression, then there is a problem with your construct.