I am trying to express and purify nanobodies from a nanobody library in E. coli. I am using a pBAD vector to which I added the pelB sequence for periplasmic localization. The nanobody is fused to YFP with a His tag.
I induce the expression with arabinose and I can see on an SDS-PAGE gel that the nanobody is being expressed (although the expression seems to be lower than for some of the other proteins that I am expressing using the same vector), but I am having trouble with the extraction and purification steps.
I have tried to extract the nanobodies using lysozyme with PMSF following a protocol that usually works for me and I have also tried the osmotic shock protocol (Article A general protocol for the generation of Nanobodies for stru...
) but the nanobodies seem to be stuck in the cell pellet even after lysis.I do not have any experience with nanobodies so maybe there is an important step in the protocol that I am missing or not doing properly. I would appreciate tips or good protocols for expressing and extracting nanobodies.
Hello.
In my opinion, you need to check if the nanobody is aggregated into inclusion body which is insoluble. After osmotic shock, you should analyze the supernatants and pellets by SDS-PAGE if the proteins were in the pellet. If the proteins were in the pellet, an additional solubilization process is needed.
Article Screening and purification of nanobodies from E. coli cultur...
Maybe I was not clear enough in my original post, but I always analyse the supernatants and pellets by SDS-PAGE after osmotic shock/lysozyme extraction and the protein is present in the pellet, I am just not sure how fix that.
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