Hi Sutapa, With regards to insect cell expression, how do you know it wasn't expressed? You didn't see a band by coomassie staining or by western?
If by staining, I'd recommend to just try expressing a limited amount through infection of a few 150mm dishes of your favorite cell line (I use High fives), and then do a quick lysis and purification over a small (200uL) column. In my experience, most of my proteins (including some big ones around 100kda) don't show up readily on a whole cell lysate, but you can actually see it and have quite a bit of it after seeing it on a column. While it's a pain, I find it far easier to get larger yields of protein from monolayer plates of insect cells vs suspension culture. Something about plates produces far happier cells = more protein. Are you using a His or GST tag for insect cells? My experience has always been use GST for insect cells, the purification is far simpler and your protein is much, much cleaner as a result. Hope it helps, P
