How to: Selenomethionine

C Larsen clarsen at bimcore.emory.edu
Thu Feb 15 16:04:50 EST 1996


I've seen you guys wondering about selenomethionine and I thought I'd
pass along the method Ive recently used to collaborate with Xtal ppl.
The structure of our protein is solved now. Below is a chapter from my 
Diss'n methods. If you use it, all I ask is a reference to Christopher
Larsen, personal communication) because this is not yet published.

I found that the stock Leahy or Hsiao media did not work for me. Not
only do you have to "kick start" the bugs on LB media, but you also
have to supplement the media with good stuff so they dont croak! Its
Ok, the mass spec says total incorporation is achieved, and the metals
dont interfere with our structure.

Im not willing to discuss any more, but felt my experience would be
helpful to anobody struggling with the same problems.

? then email me  clarsen at bimcore.emory.edu

	
C. Preparation of selenomethionyl protein
	Several attempts were made to generate crystals from purified protein
with and without substrate. Several conditions for crystallography were
tried with our collaborators involving ammonium sulfate and
polyethylene glycol water removal from the aqueous mother liquor. These
crystals were adequate, but no metal derivatives were stable. 
Satisfactory crystals useful for obtaining phase information from the
native data set were not obtained until MAD phasing with
selenomethionine labeled protein was used (Leahy et al., 1992).
	For generation of selenomethinionyl protein, selenium was incorporated
into the methionine sulfur position with a met auxotroph of E.coli.
(Hsiao, 1994; Leahy, 1992). The PET (Novagen) or pRSET (Invitrogen)
expression plasmids were grown in the gal-, met- auxotroph B834(DE3) of
the BL21 strain (Novagen) was grown on LB agar as colonies. A single
colony was inoculated into 50 ml LB media and grown overnight. The
colony was diluted into Modified M9 buffer (below) and supplemented
with selenomethionine to 50 ug/ml (Calbiochem, Lot B10321). M9 minimal
media (Maniatis, 1991) was boiled for sterility, as autoclaving
carmelized the glucose. Trace transition metals and essential cofactors
were added; solutions "O" and "V" were used according to Weber, et al
1982) 
	At an OD600nm=0.6, the cells were induced with 0.5 mM IPTG. The
protein was purified (Chapter II, Methods). Ion electrospray mass
spectrometry of the resulting 16 mgs showed an incorporation of >98% Se
at each met codon. Activity measurements showed no less than 40
Units/mg specific activity.


	Modified M9 media (Sambrook et al., 1992): 6 liters 1x M9 salts were
supplemented with 12 ml Solution "O" (8 mL HCl conc, 5 g FeCl4H2O, 184
mg CaCl2 2.H2O, 64 mg H3BO3, 40 mg MnCl2 .4H2O, 18 mg CoCl2 6.H2O, 4 mg
CuCl2 .2H20, 340 mg ZnCl2, 605 mg Na2MoO4 . 2H20, taken up to 0.5 L),
170 ml Solution "P" (1.8 M  K2HPO4 Buffer at pH 7.5), 10 ml "S"
solution (0.276 M K2SO4), 120 mL 40% glucose in ddH20, 6 mL thiamine 1
mg/ml, 10 ml "V" solution (2.2 mg biotin, 2.2 mg folic acid, 220 mg
PABA, 220 mg riboflavin, 440 mg pantothenic acid, 440 mg niacinamide,
440 mg pyridoxine HCl, 440 mg thiamine HCl, 10 uL Chloroform, taken to
1.0 L), and  6 mL "T-U" solution. The media solution was boiled for
sterility, but not autoclaved, as this caused carmelizing glucose
oxidation and metal precipitation; vitamins were sterile filtered into
the media last. Trace elements were adapted from Weber et al (1982)
Proteins: Str.Func.Genet 13:275.





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