Crystallography‎ > ‎Protocols‎ > ‎

SeMet incorporation

Link to page with consideration and example on how to choose residues for mutagenesis for SeMet incorporation.

Best protocol I used (and that worked) found here:
Protocol for SeMet incorporation by metabolic inhibition. Notice that expression has to be ok in minimal medium and best is that it is at 37C - the cells grow too slowly for lower temp. - also slightly higher temp is good since one of the Met enzymes is naturally temp sensitive. So probably would be good to step from lower than 37C in LB to higher than 37C in the M9. Notice that this is _really_ minimal M9 - there is an option in some versions to add amino-acid supplements - I think this would screw-up the inhibition effect but don't know for sure. Notice that I did not add a Ca salt at all - this is also an option in some versions of M9. Notice that the Fe salt is my own 'special ingredient' that is not a part of the basic M9 medium. I added it to give the cells plenty of chance to make cytochromes for aerobic growth. So I usually divide up 1l between four 2l baffled flasks to give really good aeration. Maybe some SeMet will get oxidized but you get reduced SeMet protein intracellularly for sure - I checked it by mass-spec. 5X stock of M9 is 30g Na2HPO4 15g KH2PO4 5g NH4Cl 2.5g NaCl in 1 litre autoclaved so a 1 litre culture was made from: 200ml this 5X M9 stock 800ml water (autoclaved) 1ml 1M MgSO4 stock (autoclaved separately but still gives a slight cloudiness on addition) 10mls 4% (w/v) glucose stock (sterile filtered) 100uls 0.5% (w/v) thiamine vitamin (sterile filtered) (Ref. Ausubel et al. Current Protocols in Molecular Biology) to which I added: 1ml FeIISO4 (sterile filtered) to which you should add: + the required antibiotic Steps for expression. 1. Overexpressing clone was in E. coli BL21(DE3) host. 2. Grown overnight in LB medium (+ antibiotic). 3. Prepare 1 litre of M9 medium from components split into 250mls for good aeration in baffled flasks 4. Temp. equilibrate to 37oC _and_ aerate pre-inoculation 5. Inoculation was 1 ml of the LB overnight culture centrifuged _gently_ (1 - 2 min at about 1300Xg in a benchtop microfuge) at room temperature and cell pellet _gently_ resuspended in approx 1ml of M9 temp equil. medium. 6. This 1 ml M9 medium with resuspended cell suspension was returned to to 250 mls culture. 7. Growth was continued until 0.3 A600. Growth is really slow - from 1/2 to 1/5th the usual growth rate for the strain in rich medium. 8. At this point solid amino-acid supplements were added to the cultures to the final concentrations given: L-Lysine 100mg/l, L-Phenylalanine 100mg/l, L-Threonine 100mg/l, L-Isoleucine 50mg/l, L-Leucine 50mg/l, L-Valine 50mg/l, And L-SelenoMethionine 50mg/l. And, yes, I did just add the solid powders - admittedly weighed out into sterile microfuge tubes. Other people who have followed my protocol tell me that they have managed to resuspend the AAs in water and filter sterilize the concentrated stock. This was taking me too long so I went for the quick and dirty approach. I did resuspend the SeMet in water for addition but did not sterilize it. I think is is not worth going for D,L SeMet even if it is cheaper. 9. After 15 min for inhibition of Met synthesis to start, the cloned protein expression was induced as usual (1 mM IPTG). 10. Growth continued for a further 6 hrs. (notice that I usually induce for only 1hr in rich medium!) Harvested as usual. 11. Purification as for native protein but a) buffers bubbled with N2, b) DTT concentration increased 5X (to 10mM final concentration) 12. SelenoMethionine incorporation into expressed and purified protein was >95% as assayed by ESI Mass Spectrometry. And no evidence for oxidation to selenoxide or selenone - well, evidence is that mass peaks are the same breadth as the native - so any oxidation is not any worse than the Met-version. 13. Crystals were grown in N2-gassed perspex boxes.