How will metals be recruited in proper coordination and how will a particular metal be selected to its target protein?
If I want to express a metalloprotein that needs two ions for its stability and if bacteria cannot recruit it (as it don't have) to the target protein,How to include those metals in the growth medium and what is the chance of its uptake into the bacteria?
What are the problems that associate with the metallo protein purification ?
Hi Santhosh
There are several published papers where researcher's have used metals in the growth media, and many reports show evidence that it helps in achieving biologically active protein.....metals have also been used during the purification procedure etc....but there may not be a standard protocol for all metallo proteins...you may have to hit and try if it is a new protein....or you may try previous protocols first is similar proteins have been mentioned in the published reports
bye
The situation depends a lot on the metal ion and the particular protein. Often it is good enough to add the metal ion to the media or the natural abundance is sufficient. Zinc for example is abundant in media and buffers. If the metal does not get incorporated spontaneously, one can add the metal ion to the medium or to the cell free extract in 100 microM to 1 mM concentrations. IF you add it to the cell free extract, one can let it incubate for half an hour. Beware that this can also precipitate your protein if you add to much metal. However, adding metal ions to the cell free extract often works much better than adding it to the purified protein. For some proteins it takes weeks or longer for a metal ion to enter the apo protein once it is purified. For other proteins, one can reconstitute an apo protein within seconds. This probably depends on the rigidity of the protein around the metal site and how much the site is buried within the protein.
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