Question

You are trying to design a protein that will be expressed in Escherichia coli and secreted outside of the cell for purification and use as a pharmaceutical. E. coli is a gram-negative cell and the protein folds after it has exited the cell. Which secretion system would work best for this project? Support your answer with evidence based on the properties of E. coli, the protein, and the secretion system

Answer 1

The exact secretion system you’ll need to use for this kind of experiment is the Type II secretion system (T2SS) since this maintains the protein in an unfolded state in cytoplasm and secretes it through a pore in the inner membrane (through Sec or Tat systems) and afterwards is secreted by the outer membrane secretins and is folded in the exterior medium conditions.

In the protein I’d like to express I would add a 5’ tag to the gene (along with a modification to fuse it to a strong promoter) according to the aminoacidic sequence that is recognized by the signal recognition particle (SRP) so the peptide can be secreted by the Sec system.