I have been trying to purify proteins using pFuse vectors which has IgG1-Fc tag for purification. The protein should be secreted out as they have signal sequence for it. But I have been facing problems in getting the proteins secreted. I established stable cell lines with zeocin selection with the empty vector and proteins of my interest.(expected mol wt of one with 85 kDa and the other with 140 kDa).
But I can get the empty vector secreted in the media, the one with 85 kDa is getting secreted very little and the 140 kDa is not getting secreted at all. But I can see them in the lysates. I harvested the conditioned media when they are confluent and did a western as a read out. I am kind of stuck now. 293T cells have been used with pFuse vectors for purifying processes.
Any help/suggestions would be appreciated.