Hi all,

I'm in the situation, where a user is adamant to have a fusion protein made which contains an extracellular domain of a membrane protein and a GST tag for the purpose of immunization and antibody generation (don't ask why ! - it's a long story). One could (and we have tried this in the past) try intracellularly in E. coli, but it has only worked one in three times. Therefore, the idea is to generate a stable line of some description with Leader-ProteinX_Extracellular-3C-cleavage site-GST and harvest the fusion protein from the supernatant. If it works, all parties will be happy. I have identified one publication from Arne Skerra's lab from 1997, where they render GST "secretable" in E. coli by mutating 3 cysteine residues. However, I haven't seen much on the subject since then and hence I ask the oracles in here ;-) - any joy with secreting GST-fusion proteins in insect or mammalian cells ?

Thansk for any input,

Svend