> Dear all > > Is there any method to check membrane protein overexpression using GFP when > the C terminus is in periplasm? My reading so far all mention that for C > terminus fusion to work, it has to be cytoplasm. > > Thank you.
Dear Theresa, Superfolder GFP (sGFP) is reported to translocate and fold into the periplasm of E. coli using a secretary signal peptide (Aronson DE et al.Traffic. 2011), suggesting it would likewise be active when fused to the C-terminus of a membrane protein. Because sGFP is no longer sensitive to upstream membrane protein integration the formation of any aggregates (inclusion bodies) are likely to be fluorescent. Nonetheless, with FSEC you should be able to quickly estimate the amount of folded material. Alternatively if your membrane protein has an Nin topology you may consider the addition of an N-terminal GFP fusion. Again, you have the drawback that you can get uncoupled translation of the N-terminal fusion resulting in "free" GFP. However, as long as you know this then one can simply isolate membranes first before estimating overexpression levels. If you have an Nout topology the N-terminal GFP may affect folding/targeting and I would probably avoid this route. One last option it to consider the addition of an extra TM segment. Jeff Abramson's group has these vectors (Hseih JM et al. Prot. Sci. 2010). Best of luck! Cheers David. Dr. David Drew Royal Society University Research Fellow Division of Molecular Biosciences Imperial College London South Kensington Campus London SW7 2AZ d.d...@imperial.ac.uk Tel: 020-7594-9624 (ext. 49624)
