Hi Hari, These vectors are indeed not commercial ones. Unfortunately, I do not have any of them any more. If I were you, I would directly ask the group where they were originally made - not sure if they are always reading the ccp4 bulletin board though. Anyways, here are some more things about CYP51s to mention so that you get a better idea about what are currently working on. Problem not only with CYP51s but P450s in general is that they are often tough to deal with. That is also one of the reasons why often research groups are specialized on these ones (or even departments!) and to be honest I have the impression that in some publications important experimental details are omitted which can make it difficult to repeat the published experiments.
List of what you might/should try: -get a special vector for CYP51 expression, e.g. the pCWori+ vector (as far as I know this vector was not fully sequenced when it was originally made, could be that nowadays different subtypes exist - from lab to lab so to speak, it is supposed that this vector should not be used for expression rates below 25 degress, but this can be questioned) - use delta-amino levulinic acid as heme precursor (not sure about other precursors, but this one is pretty small and gets ingested easily by the cells and is also metabolically not far away from the porphyrin ring, means, this one makes perfect sense!) - use a metal trace solution (including Fe2+) (not sure about reports where dALA and/or Fe2+ were omitted, maybe the expression rate was low or not every polypeptide chain incorporated the heme, see below) - most likely, you need a slow expression rate (e.g. low IPTG, low temp., low shaker speed, no buffles, Fernbach flasks really required?) - consider other additives like vitamins or even drugs that coulb bind the active site and eventually stabilize the protein (if you use such drugs already for expression experiments they should not be too pricy of course, maybe cholesterol or even estradiol could work here - I guess they should also get easily into the cells during the expression procedure) - often a rich medium like TB is used, maybe it makes also sense to try sth. with less nutrients like LB? - try differnent constructs, especially consider cutting the membrane domain (check the literature for soluble CYP51 constructs) - at least in some human P450 cases coexpression with chaperones helps a lot - if you see an empty SDS-PAGE gel after native purification also try to purify the protein in the denatured form and try a Western blot (e.g. anti-his tag) - if you get protein, do "activity tests". (Easiest is to reduce the protein with dithionite in the presence of CO (BE CAREFUL!!! USE HOOD!!!). Therefore check the absorbance spectrum e.g. between 400-500nm. If your protein is still alive, you should see an abs. max. around 420nm (oxidized form) first but which decreases over time leading to a max. around 450nm.(reduced form), be careful: drugs might shift the spectrum - see literature about changing the spin states of the heme iron) - check if +- all of your protein was able to incorporate the heme by measuring the protein conc. the conventional way and compare it to the result you got from the Omura/Sato method (JBC 1964) - every protein is different of course but if it becomes really hard for you, then you should ask yourself if there might be "highly specialized superfreaks" around which are trying (maybe already for some time) to figure out the same as you and if it is realistic to try to compete with these guys... Maybe someone else has other ideas about that? Good luck! Jan --- Hari Namboodiri <hari_nambood...@epistemebiolabs.com> schrieb am Fr, 3.9.2010: > Von: Hari Namboodiri <hari_nambood...@epistemebiolabs.com> > Betreff: [ccp4bb] Heme Proteins > An: CCP4BB@JISCMAIL.AC.UK > Datum: Freitag, 3. September, 2010 19:30 Uhr > Dear CCPeers > > I wanted to thank everyone for sharing their experiences > and giving valuable advice on CYP51 protein expression. It > seems that special vectors like pCW-Lic or pcWORi is needed > for efficient expression. I have not found any commercial > source for them though. Could anybody be willing to provide > me any of the above vectors. I would be highly obliged. > > Thanks > Hari >
