I used HEK293 cells as expression system, transiently transfected using PEI. Harvested on Day 4 or sometimes Day 5. The enzyme produced always show up as strong band in cell pellet in western blot , while there is no band in supernatant. While doing enzyme assay, this shows no activity.

I changed my expression system and used Expi293 cell line, which produces an active enzyme.

My questions:

1. Is this related to cell lines, but in past we have seen HEK293 cell line producing any other enzymes?

2. Does vector design plays any role in this?

Iram