I am interesting in purifying a periplasmic protein from E.coli. It is not membrane bound. Is there is book/review I can refer to optmise/maximise protein production.?
Are you looking to purify endogenously expressed protein or something expressed from a plasmid? What kind of periplasmic protein is this? Have you checked out the NEB T7 Shuffle system?
Its expressed from a plasmid under arabinose promoter. It doesn't have issues with disulphide bond formation because it lacks intrinsic cysteines, so NEB T7 shuffle system doesnt help. I am just looking for basic tips actually.
In that case, is there any reason you need to keep the signal peptide? Can you just express and maintain it in the cytoplasm? What have you tried (in terms of expression) to this point?
Hi! Well, it has been classic to use osmotic shock for the extraction of periplasmic proteins. Even when the extraction yields could be quite low, the purity tends to be high because no contamination with proteins from cytoplasm should occur. For the purification, you could use an approach including expanded bed adsorption (https://www.ncbi.nlm.nih.gov/pubmed/8818269) or simply, any other adsorption chromatography in the first step, that allows you to concentrate the sample after the osmotic shock. Hope this helps.
- Badges
- Science topic
- Similar topics
- Geoscience
- Asia
- India