Hi,
For the purposes of protein crystallography, bacterial cultures are
routinely grown in agitated-aerobic conditions and nothing bad happens
to the se-met proteins (reducing environment of the bacteria helps, I
bet). Once you lyse the bugs, you have to have reducing agents in your
buffers (if you're doing IMAC purification then consider 0.05% - 0.1%
BME, most other chromatographic techniques do not mind 2 mM DTT) but in
my experience degassing isn't vital. If you store selenomethionine
protein at 4C for a really long time it will get oxidized however this
process is usually paralleled by 'spoilage' of the protein due to other
factors. If you wish to oxidize Se-Met quantitatively, you could use
mild peroxide treatment.
As an extreme example in my experience, a thermophilic selenomet-labeled
protein has survived prolonged heating to 70 C (buffer contained 5 mM
DTT, but wasn't particularly well isolated from oxygen). Out of 7
seleno-methionines, only one has decomposed (quantitatively lost a
-SeCH3 fragment according to ESI-MS). When the structure was solved, all
the seleniums were found with occupancy factors close to 1 except for
the one located close to the N-terminus which was unstructured, causing
high degree of solvent exposure.
Hope it helps. If you fear oxidation, you can quickly find out how bad
things are using MS - on a reasonable sized protein, ESI-MS gives < 1 Da
accuracy which is more than sufficient to detect oxidation and Se-Met
decomposition. We always monitor our purification by MS and so far have
never run into oxidation of Se-Met proteins as long as we use reducing
agents and work reasonably fast.
If you do get oxidized selenium in low abundance, do not despair.
Firstly, there are indications that some forms of oxidized se-met are
even better for phase determination. Secondly, there is a considerable
difference in hydrophobicity of different oxidation forms of selenomet -
sometimes HIC can resolve the oxidized form from the reduced one.
Artem
Michael Witty wrote:
>> Dear All,
> my protocol for selenomet protein production calls for N2 degassed
> buffers, which will prevent oxidation of the Se-Met. Considering the fact
> that the bacteria will be grown aerobically, do we think this is a
> necessary thing? Regards, Mike.
--
|Dr. Artem Evdokimov Protein Engineering |
| NCI-Frederick Tel. (301)846-5401 |
| FAX (301)846-7148 |
|eudokima at mail.ncifcrf.gov |
|http://www.ncifcrf.gov/plague |
