Protein secretion wtih pFuse expression system in 293T cells - (Jan/13/2014 )
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.
Have you done immunofluorescence analysis to confirm appropriate trafficking / folding of these proteins? They might be stuck in the ER or Golgi hindering their secretion. Also, is the tag on the right terminus? Any tag before the signal sequence will be chopped off. Some other rare possibilities might include mutation in signal sequence / kozak sequence / abnormal stop codon introduced somewhere in the middle. Hope this helps..
There are some vectors available with improved signal sequences for secreted proteins, the difference can be massive! While its best not to mess with your natural protein, in theory the signal peptide will be cleaved upon secretion leaving the same mature protein. Oh, and as above, make sure your tag is at the C-terminus, not the N!