Re: [ccp4bb]: methionine auxotroph strain

[Phraenquex VD <loretta@scripps.edu>]

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> 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.