Help for transient expression in HEK-293 - (Aug/17/2006 )

Hello everybody,

I'v been transfecting HEK-293 cells for over 1 month using lipofectamine 2000. My recombinant protein will be expressed and secreted to the cell medium. I got the my recombinant protein, although the amount is not very high(~10 ug/ml). I also comfirmed the protein by western blot. It was expressed, however, the activity of the protein is very low.

Is there any reason that can affect the protein activity? I'v already used several batch of HEK cells from liquid nitrogen. And I checked the CD393 medium which I use to incubate the cells after transfection. It is ok.

A highly possibel reason I can think of now is mycoplasma. Because others in my lab found their primary cells couldn't grow in our incubtor. So we suspect there's mycoplasma contamination. Is it possible for mycoplasma to affect the protein expression?

Your suggestions are high appreciated!

Mycoplasm could affect protein production, infact a whole bunch of things.

Before everyone panics, confirm if there is mycoplasma or not.

can you work for a while in other lab (use their cells and incubators)?
that would be a easy answer.
How do you purifythe protein (if yes the procedure may slightly alter the protein leading to misfunctions)

Thanks for your suggestion!

I didn't purify the protein. I collected the cell medium which contains the protein. My protein is a G-CSF conjugated protein, so I use cell proliferation assay to determine the activity of the G-CSF part.

Another question:
Will antibiotic in the cell medium affect the the protein's activity?
The previous student who worked on this project mentioned that before she use antibiotic-free medium when cells express the protein. I use antibiotic-free only when I do the transfection.

Thanks!
I will send my cells to the mycoplasma test.

Does any one know a good protocol for lentivirus concentration. I am trying ultracentrifugation but still getting very low titers.