Re: [ccp4bb]:SeMet incorporation by metabolic inhibition

[Martyn Simmons <martyn@cryst.bioc.cam.ac.uk>]

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Dear All
        - although this is not directly relevant to the subject of the
discussion several people have asked for more details of of my experiences
with SeMet incorporation by metabolic inhibition.  Experience is limited
to maybe three or four examples in our lab. However in each case it was
successful in producing protein that gave crystals suitable for
successful SeMet MAD experiments. (On a CCP4 note I found SCALA, REVISE,
VECREF, and DM invaluable in my data analysis - so thanks to the authors of
those programs especially). 

To reiterate: this is just my interpretation of the method originally
worked out and described in:

VanDuyne G.D. et al. (1993) J. Mol. Biol. Vol. 229 pp105-124

to whom, I think, goes the credit for thinking and developing of this
novel and elegant approach (correct me if I am wrong). 

I found this reference originally in the excellent review by S. Doublie
'Preparation of selenomethionyl proteins for phase determination' METHODS
IN ENZYMOLOGY, 1997, Vol.276, pp.523-530
                                     regards
                                          Martyn
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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)

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.