Yes, we could just express BirA from a plasmid, but we hope to eventually use this protocol in cells that are difficult to transfect. It would be more convenient to just toss some BirA into the cell lysate as long as it efficiently adds biotin to our AviTagged protein of interest.
Hi Beth :
Biotinylation of the Avi-tag needs (BirA+Biotin+ATP). There are well-established in vitro methods for that, but none that I'm aware of for doing it in lysates. Doing it in vivo has the further advantage of no additional rounds of purification & protein loss during BirA-removal. Another option may be to make a bi-cistronic plasmid where the upstream expression is of your Avi-tagged gene-of-interest, and the downstream gene could be the BirA. This has worked well for us in BEV as well as E. coli expression systems.
Good Luck,
Ron
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