Exogenous protein expression - Problems in protein expression mammalian cells (Dec/15/2008 )
I´m trying to overexpress a structural protein in human cell lines without success.
I have tried two diferent vectors (pcDNA and lentiviral vectors), checked the transfection eficiency, tried diferent cell lines including cos, hek293T, DLD-1, HT29... and others... I have also checked the sequence of the constructs and they are ok, and it includes a Kozack sequence before the ATG.....and I had never get to overexpress this protein (I always check the overexpression by western blot, and the antibody works properly).
As you can imagine, I am completely lost at this point of my research.
I have read that sometimes the exogenous DNA can be toxic... does anybody know how to solve this problem? or how to check if that is the real problem?
I have to say that my protein is quite large, about 135KDa... has any of you get to overexpressed such a large protein?
Thank you in advance!!!
what is the cell line that you are using and the method of transfection? You mentioned HEK and other cells. It can be that your cell line is particularly resistant to the method that you are using. Maybe trying electroporation might help although the technique is quite tricky. Having expression of your protein of interest in another cell line reassures you that your sequence and vector is okay but does not tell you anything about the efficiency of transfection in the cell line that you want to work with. I hope it helps
Thank you Maria for answer me.
I used lipofectamine with my pcDNA vector and lentiviral infection with the lentiviral vector (I have my gene cloned in these two different vectors). This two methods had worked properly in other proyects with the cell lines that I am working with, so I guess that the problem is not the trasfection method. Besides, I have checked the transfection eficiency (GFP) and it works.... The thing is that I have never seen overexpression of my protein in any cell line and trying diferent transfection methods. So, in this point, I am checking if the problem is transcriptional (measuring mRNA levels) or if the protein is degradated (by proteasome inhibitors)...
I will tell you how it goes....
Thank you again for helping me!!!