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Re: [ccp4bb]: methionine auxotroph strain

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Dear All
         I agree 100% with Frank about not needing an auxotroph (and this
approach has been widely used in other labs) 
         But notice the typo that I introduced when posting this recipe
(which was the subject of a separate erratum to this bulletin board): 
it should be _100mls_ of 4% Glucose (as per the usual M9 recipe). Also I
neglected to type the line that FeSO4 is _4.2 g/l stock_
         sorry about those mistakes
         although this works with strains that are good growers and
express well at 37oC it would probably be a problem if you need to express
at lower temp with a poor growing strain. 

         remember that the yield will most likely be smaller and that the
crystallization conditions can change. For example watch out for arsenic
containing buffers (eg cacodylate). Arsenate was an additive to my
native crystallizations but it gave an marked chemical reaction with the
SeMet protein. In any case arsenic has an absorption edge near Se and it
could screw up your fluorescence scans during the MAD experiment. 

On Tue, 5 Sep 2000, Phraenquex VD wrote:

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> ***    CCP4 home page http://www.dl.ac.uk/CCP/CCP4/main.html    ***
> > Dear all,
> > Can you give me some information where I could order proper Methionine
> > Auxotroph strain for overexpressing Se-Met protein?
> > Thanks in advance,
> Dear Yuan
> If you're in E. coli, you may make your life a lot easier if you simply
> stay in your usual strain and use metabolic inhibition of the methionine
> pathway to encourage Se-Met incorporation.  This has worked in all the
> cases we tried in our lab (8+ and counting) and you save the trouble of
> having to establish expression in some temperamental strain you don't know
> at all.  
> Martyn Symmons sent out the protocoll a while ago, I attach it again.
> It really is rather easy to do.  The (or a?) reference is:
>   van Duyne, G. D., R. F. Standaert, et al. (1993). Atomic structures of
>   the human immunophilin FKBP-12 complexes with FK506 and rapamycin.
>   Journal of Molecular Biology 229(1): 105-124.B
> Good luck
> Phraenquex
> -----------------------------------------------------------
>  Protocol for SeMet incorporation by metabolic inhibition.
> Notice that expression has to be ok in minimal medium and best is that it
> is at 37oC - 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 37oC in LB to
> higher than 37oC 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
>                  1ml FeIISO4 (sterile filtered)
>                  + stock 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. equibrate to 37oC _and_ aerate pre-inoculation
> 5. Inoculatin 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 resupend 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 degassed 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.