Hi Lauren

No need to include the signal peptide.

Additionally, you can tag your protein i.e. His-tag to improve protein purification in BL-21 sys.

Hope that work well

You could try OmpA tag to bring it into outer membrane of E. coli.

The suggestion is that you should try the Saccharomyces cerevisiae system (such as: pgapz alpha vector and P. pastoris X-33 )for insertion protein of the native conformation. The signal peptide would be cleaved when located on the membrane in the mammal cells.

Hi Lauren,

Is you plan to express the OM protein in the E. coli OM? If so, you will definitely need to include a signal peptide. You can try the native SPs, but I would also consider testing an E. coli signal peptide, such as OmpA or MalE, in parallel. Another issue might be the so-called BAM signal, which is in the C-terminus of the OMPs and guides recognition by the BAM complex for insertion into the OM. Brucella is phylogenetically somewhat distant from E. coli, so the Brucella proteins might not be recognised efficiently by the E. coli BAM. You will just have to try. If yo don't get good insertion (which you can assay by heat modifiability), you could try a DsbA signal peptide which works a bit slower and might help. You could also try some strains we made for improving heterologous OMP production (see Article A New Strain Collection for Improved Expression of Outer Mem...

I'm not very familiar with the SUMO system; where are you going to place the tag? A large tag like SUMO might also have an effect on OM integration. You might consider trying smaller epitope tags (His, Strep or similar).

Hi Lauren,

You need to add a signal peptide corresponding to the expression system in front of the target protein. In addition, the SUMO tag is relatively large and is not recommended to be at the same end as the signal peptide. I remember that the E. coli expression system has the expression vector suitable for secreting proteins, you can try it.

If you need your protein secreted to matrix, which should have a signal peptide.

Some vectors have own signal peptides, in that case, you don't need to need the target protein's signal peptide.

It is difficult to predict whether the Brucella signal peptide will function in E. coli or not, and how it would behave. Since this is a membrane protein, most likely if you express it in E. coli without any signal then you will end up with insoluble protein probably as an inclusion body. If you want to try to express and have it insert in the E. coli OM then you do need a signal peptide. Using an E. coli signal, like OmpA should certainly work, using the Brucella one may or may not work. If I were doing the experiment I would likely try it both ways to see which works best. It is only one additional primer and one additional PCR reaction and cloning.

I agree with a number of the answers above, in particular with Jack. However, these all suppose that you want / need to express the full protein. If that is the case then as several have said it would need to have a signal and be directed to the E.coli membrane, and Brucella / E.coli signals could both be tried. You'd then need to perform membrane preps and presumably isolate the protein in a detergent of some sort. You might even try using SMALPs (see: http://www.smalp.net/protocols.html also worth googling as there are lots of potentially useful refs out there).

It may be though, that for your purposes (ELISA) you'll want soluble protein? If that is the case perhaps you should consider generating constructs that will express just the extracellular domain. You could use structural information / prediction to identify the domain of interest, then clone that into your preferred expression system. There are numerous tools to help with this e.g. see here: https://www.expasy.org/proteomics/protein_structure. You would not need leaders in this case, as soluble cytoplasmic expression ought to work (depending on the sequence). If you go this route ask your local Structural Biologist for help determining the domain and designing your constructs.

Good luck.