| Hary, Delta-aminolevulinic acid is the classical heme (heme b) precursor for your experiments. As far as I know, the synthesis of porphyrin goes very quickly in E. coli, since I tried it once with a bacterial P450 and got a fat overexpression band after ~2hrs in the lysate. Also the photometric experiments worked well afterwards, means the protein also contained the porphyrin. However, I was also working on a CYP51 and important in this case was to make sure that the cells do NOT get so much air during shaking. That means e.g. low shaker speed, no buffles etc. Temperature should be low, but this also depends on your vector. Do you use e.g. pCWori+ - pET vectors do often not work for CYP51s - ? Sometimes, there are other additives rather than dALA used like thymine etc. Moreover, for these P450 expressions, often Fernbach flasks are taken (they are a little bit wider on the bottom than the Erlenmeyer flasks). I wonder if this really makes sense for the CYP51 expressions though, because the idea of these bottles is to INCREASE the interface of bacterial medium and air.... HTH Jan --- Hari Namboodiri <[log in to unmask]> schrieb am Do, 26.8.2010:
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